Separation and Characterization of the Related Substances of Bedaquiline Fumarate Using HPLC and Spectral Methods.

The study focuses on isolating and characterizing the potential degradation products (DPs) and impurities of Bedaquiline fumarate in bulk drug form. A stability-indicating related substance method was developed and validated using high-performance liquid chromatography. Agilent Poroshell 120EC- C18 (150 mm × 4.6 mm, 2.7 µm) column showed an optimum separation of 10 analytes. The mobile phase contained 0.05% trifluoroacetic acid, acetonitrile and methanol in a time gradient mode. Bedaquiline fumarate was susceptible to acid hydrolysis, oxidation, base hydrolysis and showed three potential DPs, including DP-1, DP-2 and Impurity-8. Degradation products, DP-1 and DP-2, were isolated and characterized by ESI-MS, 1H, NMR and 13C NMR spectroscopy. The developed method was validated according to ICH tripartite guidelines and showed adequate specificity, accuracy and linearity. The limit of detection and limit of quantitation were 0.05 and 0.15 µg/mL, respectively.

Isolation and characterization of thermal degradation impurity in brimonidine tartrate by HPLC, LC-MS/MS, and 2DNMR.

One potential unknown impurity was detected during the analysis of stability batches of brimonidine tartrate (BMT) in the level ranging from 0.03 % to 0.06 % by high-performance liquid chromatography (HPLC). Based on the liquid chromatography-mass spectrophotometry (LC-MS) analysis, the unknown impurity structure was presumed as 3,6,11,13,16-pentaazatetracyclo [8.6.0.0²,7.0¹²,¹6] hexadeca-1,3,5,7,9,12-hexaene. The proposed structure was elucidated, after its isolation using preparative liquid chromatography from the impurity enriched reaction crude sample, using analytical applications such as 1D NMR (1H, 13C and DEPT-135), 2D NMR (HMBC and COSY), high-resolution mass spectrometry (HRMS) and infrared spectroscopy (IR). The unknown impurity was prepared from brimonidine by following Ullman coupling reaction in the presence of CuBr2 in gram scale with optimum purity to use further in analytical developments. The identification, structural elucidation and synthesis of unknown degradation impurity such as BMT-cyclized impurity, and HPLC method validation were reported for the first time in this paper.

Identification and characterization of Prothionamide degradation impurities by mass spectrometry, NMR spectroscopy, and ultra high performance liquid chromatography method development.

Stability-indicating and liquid chromatography-mass spectrometry compatible ultra high performance liquid chromatography method was developed for the degradation and drug substances related impurities of Prothionamide. Forced degradation of Prothionamide was carried out under acidic, basic, thermal, oxidative, and photolytic stress conditions. The impurities separation was achieved on Acquity UPLC BEH-C18 (50 mm × 2.1 mm, 1.7 µm) with the mobile phase of 10 mm ammonium acetate pH 6.0 and Acetonitrile in a time gradient mode. Related substances by ultra-performance liquid chromatography method was validated according to ICH tripartite guidelines. Degradation products were isolated by Column chromatography and characterized by liquid chromatography-mass spectrometry, 1 H, and 13 C nuclear magnetic resonance spectroscopy. The developed related substances method showed adequate specificity, sensitivity, accuracy, linearity (0.4-1.5 µg/mL), precision, and robustness in line with ICH tripartite guidelines for validation of analytical procedures. Limits of detection and quantitation were 0.1 and 0.4 µg/mL, respectively, for Prothionamide and all the impurities. The method was found to be linear with a correlation coefficient > 0.99, precise (%RSD < 5.0), robust and accurate (%recovery 85-115%).

Identification and structural elucidation of two process impurities and stress degradants in darifenacin hydrobromide active pharmaceutical ingredient by LC-ESI/MS(n).

The present study describes the identification and characterization of two process impurities and major stress degradants in darifenacin hydrobromide using high performance liquid chromatography (HPLC) analysis. Forced degradation studies confirmed that the drug substance was stable under acidic, alkaline, aqueous hydrolysis, thermal and photolytic conditions and susceptible only to oxidative degradation. Impurities were identified using liquid chromatography coupled with ion trap mass spectrometry (LC-MS/MS(n)). Proposed structures were unambiguously confirmed by synthesis followed by characterization using nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR) and elemental analysis (EA). Based on the spectroscopic, spectrometric and elemental analysis data, the unknown impurities were characterized as 2-{1-[2-(2,3-dihydrobenzofuran-5-yl)-2-oxo-ethyl]-pyrrolidin-3-yl}-2,2-diphenylacetamide (Imp-A), 2-[1-(2-benzofuran-5-yl-ethyl)-pyrrolidin-3-yl]-2,2-diphenylacetamide (Imp-B), 2-{1-[2-(2,3-dihydrobenzofuran-5-yl)-ethyl]-1-oxy-pyrrolidin-3-yl}-2,2-diphenylacetamide (Imp-C) and 2-{1-[2-(7-bromo-2,3-dihydrobenzofuran-5-yl)-ethyl]-pyrrolidin-3-yl}-2,2-diphenylacetamide (Imp-D). Plausible mechanisms for the formation and control of these impurities have also been proposed. The method was validated as per regulatory guidelines to demonstrate specificity, sensitivity, linearity, precision, accuracy and the stability-indicating nature. Regression analysis showed a correlation coefficient value greater than 0.99 for darifenacin hydrobromide and its impurities. The accuracy of the method was established based on the recovery obtained between 86.6 and 106.7% for all impurities.

A stability indicating simultaneous dual wavelength UV-HPLC method for the determination of potential impurities in fampridine active pharmaceutical ingredient.

A novel, sensitive, stability indicating simultaneous dual wavelength reverse phase UV-HPLC method has been developed for the quantitative determination of potential impurities of fampridine active pharmaceutical ingredient. Efficient chromatographic separation was achieved on a C18 stationary phase in gradient mode and quantitation by ultraviolet dual wavelength detection. The method was validated according to ICH guidelines with respect to specificity, precision, linearity and accuracy. Regression analysis showed correlation coefficient value greater than 0.999 for fampridine and its seven impurities. Detection limit as low as 0.003% was achieved for fampridine N-oxide and 0.01% for other impurities. Accuracy of the method was established based on the recovery obtained between 93.3% and 110.0% for all impurities. The method was found to be specific, selective to the degradation products and robust. Peak purity analysis by PDA detector confirmed the specificity of the method. Major degradation of the drug substance was found to occur under oxidative stress conditions to form fampridine N-oxide.

Highly efficient, selective, sensitive and stability indicating RP-HPLC-UV method for the quantitative determination of potential impurities and characterization of four novel impurities in eslicarbazepine acetate active pharmaceutical ingredient by LC/ESI-IT/MS/MS.

A novel, sensitive, selective and stability indicating LC-UV method was developed for the determination of potential impurities of eslicarbazepine acetate. High performance liquid chromatographic investigation of eslicarbazepine acetate laboratory sample revealed the presence of several impurities. Three impurities were characterized rapidly and four impurities were found to be unknown. The unknown impurities were identified by liquid chromatography coupled with electrospray ionization, ion trap mass spectrometry (LC/ESI-IT/MS/MS). Structural confirmation of these impurities was unambiguously carried out by synthesis followed by characterization using nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (FT-IR) and mass spectrometry (MS). Based on the spectroscopic, spectrometric and elemental analysis data unknown impurities were characterized as 5-acetyl-5,11-dihydro-10H-dibenzo [b,f]azepin-10-one, N-acetyl-5H-dibenzo[b,f]azepine-5-carboxamide, 5-acetyl-10,11-dihydro-5H-dibenzo[b,f]azepin-10-yl acetate and 5-acetyl-5H-dibenzo[b,f]azepin-10-yl acetate. The newly developed LC-UV method was validated according to ICH guidelines considering eleven potential impurities and four new impurities to demonstrate specificity, precision, linearity, accuracy and stability indicating nature of the method. The newly developed method was found to be highly efficient, selective, sensitive and stability indicating. A plausible pathway for the formation of four new impurities is proposed.

Identification, characterization and quantification of new impurities by LC-ESI/MS/MS and LC-UV methods in rivastigmine tartrate active pharmaceutical ingredient.

Six impurities were detected at trace level in rivastigmine tartrate drug substance by a newly developed high performance liquid chromatography method. Three impurities were characterized rapidly and three impurities were found to be unknown. The unknown impurities were enriched and identified with a combination of semi-preparative HPLC and LC/MS/MS techniques. Proposed structures were further confirmed by characterization using NMR, FT-IR, and EA techniques of impurity standards. Based on the spectroscopic, spectrometric and elemental analysis data unknown impurities were characterized as 3-[1-(dimethylamino)ethyl]phenyl N-ethyl-N-methyl carbamate N-oxide, ethyl-methyl-carbamic acid 4-(1-dimethylamino-ethyl)-phenyl ester and ethyl-methyl-carbamic acid 2-(1-dimethylamino-ethyl)-phenyl ester. A plausible mechanism for the formation of these impurities is also proposed. The method was validated according to ICH guidelines for fourteen impurities to demonstrate specificity, precision, linearity, accuracy and stability indicating nature of the method. Regression analysis showed correlation coefficient value greater than 0.999 for rivastigmine tartrate and its impurities. Accuracy of the method was established based on the recovery obtained between 93.41 and 113.33% for all impurities.