Archana Sahu

A critical review on the use of modern sophisticated hyphenated tools in the characterization of impurities and degradation products

With ever increasing regulatory and compendial stringency on the control of impurities (IMPs) and degradation products (DPs) (including genotoxic impurities) in drug substances and finished pharmaceutical formulations, a profound emphasis is being paid on their characterization and analysis at trace levels. Fortunately, there have been parallel tremendous advancements in the instrumental techniques that allow rapid characterization of IMPs and/or DPs at the prescribed levels of ∼0.1%. With this, there is perceptible shift from conventional protocol of isolation and spectral analysis to on-line analysis using modern sophisticated hyphenated tools, like GC-MS, LC-MS, CE-MS, SFC-MS, LC-NMR, CE-NMR, LC-FTIR, etc. These are already being extensively used by industry and also there is tremendous increase in publications in the literature involving their use. This write-up critically reviews the literature for application of hyphenated tools in impurity and degradation product profiling of small molecules. A brief mention is made on possible pitfalls in the experimentation and data interpretation. Appropriate strategies are proposed, following which one can obtain unambiguous characterization of the unidentified IMPs and/or DPs.

Characterization of stress degradation products of benazepril by using sophisticated hyphenated techniques

Benazepril, an anti-hypertensive drug, was subjected to forced degradation studies. The drug was unstable under hydrolytic conditions, yielding benazeprilat, which is a known major degradation product (DP) and an active metabolite. It also underwent photochemical degradation in acid and neutral pH conditions, resulting in multiple minor DPs. The products were separated on a reversed phase (C18) column in a gradient mode, and subjected to LC-MS and LC-NMR studies. Initially, comprehensive mass fragmentation pathway of the drug was established through support of high resolution mass spectrometric (HR-MS) and multi stage tandem mass spectrometric (MS(n)) data. The DPs were also subjected to LC-MS/TOF studies to obtain their accurate masses. Along with, on-line H/D exchange data were obtained to ascertain the number of exchangeable hydrogens in each molecule. LC-(1)H NMR and LC-2DNMR data were additionally acquired in a fraction loop mode. The whole information was successfully employed for the characterization of all the DPs. A complete degradation pathway of the drug was also established.

ICH guidance in practice: Degradation behaviour of oseltamivir phosphate under stress conditions

Oseltamivir phosphate was subjected to stress degradation conditions prescribed by ICH guideline Q1A (R2). A total of five degradation products (Os I to Os V) were generated under hydrolytic (acid and alkaline) stress conditions. Their unambiguous structural elucidation was carried out using LC-MS, LC-NMR and HR-NMR data. First, accurate masses of Os I, Os II, Os IV and Os V were determined by LC-MS/TOF. Subsequently, (1)H and COSY NMR studies were carried on the drug and these four degradation products using LC-NMR. The structure of Os III was elucidated after preparative isolation and purification, followed by MS/TOF and HR-NMR studies. The degradation products, Os II, Os IV and Os V were characterized as 4-acetamido-5-amino-3-(pentan-3-yloxy)cyclohex-1-ene carboxylic acid, 4,5-diamino-3-(pentan-3-yloxy)cyclohex-1-ene carboxylic acid and ethyl 4,5-diamino-3-(pentan-3-yloxy)cyclohex-1-ene carboxylate, respectively. Os I and Os III were identified as positional isomers of Os II and the drug, respectively, involving N,N-acyl migration from 4-amino to 5-amino position in the ring. Two degradation products (Os IV and Os V) were found to be new and previously unreported. The degradation pathway for all five was outlined and justified mechanistically. In silico toxicity of the drug and degradation products was also assessed using TOPKAT and DEREK software and compared.

Stability behaviour of antiretroviral drugs and their combinations. 2: Characterization of interaction products of lamivudine and tenofovir disoproxil fumarate by mass and NMR spectrometry.

This study focused on drug-drug interaction behaviour among lamivudine (3TC) and tenofovir disoproxil fumarate (TDF), the two anti-retroviral drugs. Apart from pre-known degradation products of individual drugs, a total of twelve interaction products were detected by high performance liquid chromatography (HPLC) using a C18 column as the stationary phase, and methanol and ammonium formate in gradient mode as the mobile phase. The same HPLC method was employed for liquid chromatography-high resolution mass spectrometry (LC-HRMS) and liquid chromatography-multi stage mass spectrometry (LC-MS(n)). For the characterization of interaction products, stability samples were subjected to LC-HRMS, LC-MS(n) and online hydrogen/deuterium exchange studies. Two isomeric interaction products were isolated and subjected to 1D and 2D nuclear magnetic resonance (NMR) studies. The collated information was utilized for the characterization of all twelve interaction products of the two drugs. Pathway of their formation was also outlined.

Stability behaviour of antiretroviral drugs and their combinations. 5: Characterization of novel degradation products of abacavir sulfate by mass and nuclear magnetic resonance spectrometry

HIGHLIGHTSSolution and solid state stress studies conducted on abacavir sulfate.A total of eleven degradation products detected by HPLC and characterized by mass tools.Seven degradation products isolated and structures confirmed by NMR studies.Fragmentation pattern of all degradation products along with the drug established.Pathway of formation of all degradation products outlined. ABSTRACT In the present study, degradation behaviour of abacavir sulfate was evaluated in solution and solid stress conditions. Solution state studies resulted in formation of eleven degradation products; of which two were also formed on solid stress. The same were separated by high performance liquid chromatography. They were characterized using liquid chromatography‐high resolution mass spectrometry, liquid chromatography‐multistage mass spectrometry and hydrogen/deuterium exchange mass spectrometry data. Additionally, seven degradation products were isolated and subjected to 1D and 2D nuclear magnetic resonance studies for their structural confirmation.

Study of the forced degradation behavior of prasugrel hydrochloride by Liquid Chromatography with Mass spectrometry and liquid chromatography with NMR detection and prediction of the toxicity of the characterized degradation products

Prasugrel was subjected to forced degradation studies under conditions of hydrolysis (acid, base, and neutral), photolysis, oxidation, and thermal stress. The drug showed liability in hydrolytic as well as oxidative conditions, resulting in a total of four degradation products. In order to characterize the latter, initially mass fragmentation pathway of the drug was established with the help of mass spectrometry/time-of-flight, multiple stage mass spectrometry and hydrogen/deuterium exchange data. The degradation products were then separated on a C18 column using a stability-indicating volatile buffer method, which was later extended to liquid chromatography-mass spectrometry studies. The latter highlighted that three degradation products had the same molecular mass, while one was different. To characterize all, their mass fragmentation pathways were established in the same manner as the drug. Subsequently, liquid chromatography-nuclear magnetic resonance (NMR) spectroscopy data were collected. Proton and correlation liquid chromatography with NMR spectroscopy studies highlighted existence of diastereomeric behavior in one pair of degradation products. Lastly, toxicity prediction by computer-assisted technology (TOPKAT) and deductive estimation of risk from existing knowledge (DEREK) software were employed to assess in silico toxicity of the characterized degradation products.

Use of LC–MS/TOF, LC–MSn, NMR and LC–NMR in characterization of stress degradation products: Application to cilazapril

Forced degradation studies on cilazapril were carried out according to ICH and WHO guidelines. Significant degradation of the drug was observed in acid and base conditions, resulting primarily in cilazaprilat. In neutral condition, five degradation products were formed, while under oxidative condition, two degradation products were generated. In total, seven degradation products were formed, which were separated on an Inertsil C-18 column using a stability-indicating HPLC method. Structure elucidation of the degradation products was done by using sophisticated and hyphenated tools like, LC-MS/TOF, LC-MS(n), on-line H/D exchange, LC-NMR and NMR. Initially, comprehensive mass fragmentation pathway of the drug was laid down. Critical comparison of mass fragmentation pathways of the drug and its hydrolytic degradation products allowed structure characterization of the latter. 1D and 2D proton LC-NMR studies further confirmed the proposed structures of hydrolytic degradation products. The oxidative degradation products could not be characterized using LC-MS and LC-NMR tools. Hence, these degradation products were isolated using preparative HPLC and extensive 1D ((1)H, (13)C, DEPT) and 2D (COSY, TOCSY, HETCOR and HMBC) NMR studies were performed to ascertain their structures. Finally, degradation pathways and mechanisms of degradation of the drug were outlined.

Quantitation of memantine hydrochloride bulk drug and its tablet formulation using proton nuclear magnetic resonance spectrometry

The use of quantitative nuclear magnetic resonance spectrometry for the determination of non‐UV active memantine hydrochloride with relative simplicity and precision has been demonstrated in this study. The method was developed on a 500 MHz NMR instrument and was applied to determination of the drug in a tablet formulation. The analysis was performed by taking caffeine as an internal standard and D2O as the NMR solvent. The signal of methyl protons of memantine hydrochloride appeared at 0.75 ppm (singlet) relative to the signal of caffeine (internal standard) at 3.13 ppm (singlet). The method was found to be linear (r2 = 0.9989) in the drug concentration range of 0.025 to 0.80 mg/ml. The maximum relative standard deviation for accuracy and precision was <2. The limits of detection and quantification were 0.04 and 0.11 mg/ml, respectively. The robustness of the method was revealed by changing nine different parameters. The deviation for each parameter was also within the acceptable limits. The study highlighted possibility of direct determination of memantine hydrochloride in pure form and in its marketed tablet formulation by the use of quantitative NMR, without the need of derivatization, as is the requirement in HPLC studies. Copyright

Stability behaviour of antiretroviral drugs and their combinations. 8: Characterization and in-silico toxicity prediction of degradation products of efavirenz

Graphical abstract Figure. No Caption available. HighlightsSolution state stress studies were conducted on efavirenz.Comprehensive mass fragmentation pattern of the drug was outlined.Twelve degradation products detected by HPLC and identified using LC‐HRMS and NMR studies.Degradation pathway of the efavirenz was established.In‐silico toxicity of all the degradation products was evaluated by TOPKAT. ABSTRACT Efavirenz (EFV), an antiretroviral drug, was evaluated for its degradation behaviour in solution state. A total of twelve degradation products were detected on high performance liquid chromatography (HPLC) analyses. Initially, comprehensive mass fragmentation pattern of the drug was established by direct injection and collection of high resolution mass spectrometry (HRMS) and multi‐stage tandem mass spectrometry (MSn) data. Subsequently, LC‐HRMS studies were carried on the stability samples containing the degradation products. Eleven degradation products were isolated and subjected to 1D and 2D nuclear magnetic resonance (NMR) studies for their structural confirmation. The collated information was utilized for the characterization of all the degradation products and hence in outlining the comprehensive degradation pathway of the drug. In‐silico toxicity of the degradation products was evaluated by TOPKAT analyses.

Stability behaviour of antiretroviral drugs and their combinations. 6: evidence of formation of potentially toxic degradation products of zidovudine under hydrolytic and photolytic conditions

This study explored the comprehensive degradation behaviour of zidovudine (ZDV) under solution and solid stress conditions. In total, nine degradation products were detected by high performance liquid chromatography (HPLC). The same were tentatively characterized with the help of high resolution and multistage mass spectrometry. Among them, five degradation products were also enriched and isolated with the help of semi-preparative HPLC and subjected to 1D and 2D nuclear magnetic resonance and/or infrared spectrometric studies to confirm their structures. The characterization of all the degradation products helped in outlining the comprehensive degradation pathway of ZDV. A significant finding was the formation of 3′-amino-3′-deoxythymidine (AMT) upon base hydrolysis as well as photolysis of the drug. This product is a known catabolite of the drug with a high degree of toxicity. Also, a few other degradation products formed during the study were predicted to have potential toxicity.