Molecular insight into atypical instability behavior of fixed-dose combination containing amlodipine besylate and losartan potassium.

Combination therapy with the use of fixed-dose combinations (FDCs) is evincing increasing interest of prescribers, manufacturers and even regulators, evidently due to the primary benefit of improved patient compliance. However, owing to potential of drug-drug interaction, FDCs require closer scrutiny with respect to their physical and chemical stability. Accordingly, the purpose of the present study was to explore stability behavior of a popular antihypertensive combination of amlodipine besylate (AML) and losartan potassium (LST). Physical mixtures of the two drugs and multiple marketed formulations were stored under accelerated conditions of temperature and humidity (40°C/75% RH) in a stability chamber and samples were withdrawn after 1 and 3 months. The physical changes were observed visibly, while chemical changes were monitored by HPLC employing a method that could separate the two drugs and all other components present. The combination revealed strong physical instability and also chemical degradation of AML in the presence of LST. Interestingly, three isomeric interaction products of AML were formed in the combination, which otherwise were reported in the literature to be generated on exposure of AML free base above its melting point. The same unusual products were even formed when multiple marketed FDCs were stored under accelerated conditions outside their storage packs. However, these were absent when AML alone was stored in the same studied conditions. Therefore, reasons for physical and chemical incompatibility and the mechanism of degradation of AML in the presence of LST were duly explored at the molecular level. The outcomes of the study are expected to help in development of stable FDCs of the two drugs.

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.

Explanation through density functional theory of the unanticipated loss of CO2 and differences in mass fragmentation profiles of ritonavir and its rCYP3A4-mediated metabolites.

In the present study, the metabolism of ritonavir was explored in the presence of rCYP3A4 using a well-established strategy involving liquid chromatography-mass spectrometry (LC-MS) tools. A total of six metabolites were formed, of which two were new, not reported earlier as CYP3A4-mediated metabolites. During LC-MS studies, ritonavir was found to fragment through six principal pathways, many of which involved neutral loss of CO2, as indicated through 44-Da difference between masses of the precursors and the product ions. This was unusual as the drug and the precursors were devoid of a terminal carboxylic acid group. Apart from the neutral loss of CO2, marked differences were also observed among the fragmentation pathways of the drug and its metabolites having intact N-methyl moiety as compared to those lacking N-methyl moiety. These unusual fragmentation behaviours were successfully explained through energy distribution profiles by application of the density functional theory.

Critical practical aspects in the application of liquid chromatography-mass spectrometric studies for the characterization of impurities and degradation products.

Liquid chromatography-mass spectrometry (LC-MS) is considered today as a mainstay tool for the structure characterization of minor components like impurities (IMPs) and degradation products (DPs) in drug substances and products. A multi-step systematic strategy for the purpose involves high resolution mass and multi-stage mass studies on both the drug and IMPs/DPs, followed by comparison of their fragmentation profiles. Its successful application requires consideration of many practical aspects at each step. The same are critically discussed in this review.

Characterization of a new degradation product of nifedipine formed on catalysis by atenolol: A typical case of alteration of degradation pathway of one drug by another.

An increasing interest is being shown throughout the world on the use of fixed-dose combinations of drugs in the therapy of select diseases, like cardiovascular diseases, due to their multiple advantages. Though the main criterion for combining drugs in a single dosage form is the rationale, but consideration like stability of formulation is equally important, due to an added aspect of drug-drug interaction. The objective of this study was to evaluate interaction among the drugs in an antihypertensive combination of nifedipine and atenolol. Nifedipine is a known light sensitive drug, which degrades via intra-molecular mechanisms to nitro- and nitroso-pyridine analogs, along with a few minor secondary products that are formed through inter-molecular interactions amongst primary degradation products and their intermediates. Atenolol is reasonably stable weakly basic drug that is mainly hydrolyzed at acetamide terminal amide moiety to its corresponding carboxylic acid. To the best of our knowledge, there is no known information on chemical compatibility among the two drugs. The present study involved subjecting of nifedipine, atenolol and their combination to a variety of accelerated and stress conditions. HPLC studies revealed formation of a new product in the mixture of two drugs (∼2%), which was also generated from nifedipine alone, but at trace levels (<0.1%). The product was isolated by preparative chromatography and subjected to indepth studies for its characterization. Ultra-violet, FT-IR, mass spectrometric and nuclear magnetic resonance spectroscopic studies highlighted that the principal photo-degradation pathway of nifedipine was modified and diverted in the presence of atenolol. To verify the same, a study was conducted employing two other ß-blockers with similar structures to atenolol, and the same product was formed in relatively higher quantity therein also. The new product is postulated to be produced as a result of rearrangement of hydroxylamine intermediate, known to be involved in the generation of nitro- and nitroso-pyridine photo-degradation products of nifedipine.

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.

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.

Stress degradation studies on lornoxicam using LC, LC-MS/TOF and LC-MSn.

Lornoxicam was subjected to forced degradation studies under hydrolytic (acidic, basic and neutral), oxidative, photolytic and thermal stress conditions, as defined under ICH guideline Q1A (R2). The drug degraded significantly in hydrolytic, oxidative and photoneutral conditions, leading to the formation of eight degradation products in total. It was stable on exposure to light and dry heat in the solid state. The stressed samples in which degradation was observed were mixed together and used to develop a stability-indicating HPLC method wherein degradation products were separated from the drug and also from each other. To characterize the degradation products, a complete mass fragmentation pathway of the drug was first established with the help of MS/TOF, MS(n) and H/D exchange mass studies. The same was followed by LC-MS/TOF and on-line H/D exchange experiments on the degradation products. The degradation pathway of the drug was outlined, justified by the mechanisms of formation of the degradation products.