Isolation and characterization of degradation products of moxidectin using LC, LTQ FT-MS, H/D exchange and NMR.

This study aimed to evaluate the degradation profile and pathways, and identify unknown impurities of moxidectin under stress conditions. During the experiments, moxidectin samples were stressed using acid, alkali, heat and oxidation, and chromatographic profiles were compared with known impurities given in European Pharmacopeia (EP) monograph. Moxidectin has shown good stability under heat, while reaction with alkali produced 2-epi and ∆2,3 isomers (impurities D and E in EP) by characteristic reactions of the oxahydrindene (hexahydrobenzofuran) portion of the macrocyclic lactone. Two new, previously unreported, unknown degradation products, i.e. impurity 1 and impurity 2, detected after acid hydrolysis of moxidectin (impurity 2 was also observed to a lesser extent after oxidation), were isolated from sample matrices and identified using liquid chromatography, NMR, high-resolution FT-ICR MS, and hydrogen/deuterium exchange studies. FTMS analysis showed accurate mass of molecular ion peaks for moxidectin at m/z 640.38412, impurity 1 at m/z 656.37952 and impurity 2 at m/z 611.35684, giving rise to daughter ions traceable up to the seventh levels of MS(n) experiments and supporting the proposed structures. Both unknown impurities along with moxidectin were fully characterized by (1)H, (13)C, 1D HMBC and 2D (NOESY, COSY and HSQC) NMR experiments. The interpretation of experimental data positively identified impurity 1 as 3,4-epoxy-moxidectin and impurity 2 as 23-keto-nemadectin. The identification of new impurities and correlation of their chromatographic profiles with the EP method is very useful to establish the stability profile of moxidectin and its preparations, as well as add value to the forthcoming moxidectin finished product European Pharmacopeia monographs.

An overview on chemical derivatization and stability aspects of selected avermectin derivatives.

Naturally occurring avermectins (AVMs) and its derivatives are potent endectocide compounds, well-known for their novel mode of action against a broad range of nematode and anthropod animal parasites. In this review, chemical and pharmaceutical aspects of AVM derivatives are described including stability, synthetic and purification processes, impurities and degradation pathways, and subsequent suggestions are made to improve the chemical stability. It has been found out that unique structure of AVM molecules and presence of labile groups facilitated the derivatization of AVM into various compounds showing strong anthelmintic activity. However, the same unique structure is also responsible for labile nature related to sensitive stability profile of molecules. AVMs are found to be unstable in acidic and alkaline conditions. In addition, these compounds are sensitive to strong light, and subsequently presence of photo-isomer in animals treated topically with AVM product is well known. The pharmacoepial recommendations for addition of antioxidant into drug substance, as well as its products, arises from the fact that AVM are very sensitive to oxidation. Formations of solvates, salts, epoxides, reduction of double bonds and developing liquid formulation around pH 6.2, were some chemical approaches used to retard the degradation in AVM. This coherent review will contribute towards the better understanding of the correlation of chemical processes, stability profile and biological activity; therefore, it will help to design the shelf-life stable formulations containing AVMs.

Selective separation and characterisation of stress degradation products and process impurities of prucalopride succinate by LC-QTOF-MS/MS.

The present study reports the degradation behaviour of a new prokinetic agent, Prucalopride succinate, under various stress conditions as per International Conference on Harmonization guidelines (ICH, Q1A (R2)). The investigation involved monitoring decomposition of the drug under hydrolytic (acidic, basic and neutral), oxidative, photolytic and thermal stress conditions followed by characterization of the degradation products (DPs) and process related impurities (IMPs). A rapid, precise, accurate and robust reverse phase high performance liquid chromatography (RP-HPLC) method has been developed involving mobile phase of 20mM ammonium bicarbonate buffer and acetonitrile: methanol (80:20v/v) on a Waters Xbridge-C8 (150mm×4.6mm i.d., 3.5µm) column using gradient elution. The drug was found to be degraded in hydrolytic (acidic) and oxidative conditions, whereas it was stable under basic and neutral hydrolytic, photolytic and thermal stress conditions. The method was extended to LC-ESI-QTOF-MS/MS for the structural characterization of DPs and process related IMPs. Structural characterization was carried out based on the generated molecular formula of DPs and its fragment ions. It has been observed that two major DPs were formed under each acid hydrolysis and oxidative stress conditions. The most probable mechanisms involved in the formation of DPs were also proposed. Finally, the method was validated in the term of specificity, linearity, accuracy, precision, and robustness as per ICH guidelines, Q2 (R1).

The novel acid degradation products of losartan: Isolation and characterization using Q-TOF, 2D-NMR and FTIR.

Forced degradation of losartan potassium in acidic condition resulted into three potential unknown impurities. These unknown degradation products marked as LD-I, LD-II and LD-III were analyzed using a new reverse-phase high performance liquid chromatography (HPLC), eluting at 3.63, 3.73 and 3.91 relative retention times with respect to losartan potassium (LOS) peak. All three were isolated from reaction mass using preparative HPLC and their structures were elucidated using LC-MS/MS, multidimensional NMR and FTIR spectroscopic techniques, as 5(2),11(2)-dibutyl-5(4),11(4)-dichloro-1(1)H,5(1)H,7(1)H,11(1)H-1(5,1),7(1,5)-ditetrazola-5,11(1,5)-diimidazola-2,8(1,2),3,9(1,4)-tetrabenzenacyclododecaphane,(Z)-5(2),11(2)-dibutyl-5(4),11(4)-dichloro-1(1)H,5(1)H,7(2)H,11(1)H-1(5,1),7(2,5)-ditetrazola-5,11(1,5)-diimidazola-2,8(1,2),3,9(1,4)-tetrabenzenacyclododecaphane, and 5(2),11(2)-dibutyl-5(4),11(4)-dichloro-1(2)H,5(1)H,7(2)H,11(1)H-1(5,2),7(2,5)-ditetrazola-5,11(1,5)-diimidazola-2,8(1,2),3,9(1,4)-tetrabenzenacyclododecaphane, respectively. To best of our knowledge, all three degradation products are novel impurities which are not discussed at any form of publication yet.

A novel UV degradation product of Ebastine: isolation and characterization using Q-TOF, NMR, IR and computational chemistry.

Forced degradation of Ebastine (1-(4-(1,1-dimethylethyl)phenyl)-4-(4-(diphenylmethoxy) piperidin-1-yl)butan-1-one) drug substance in ultraviolet light condition resulted into an unknown significant degradation product. This degradation product was analyzed using a newly developed reverse-phase HPLC, where it was eluted at 2.73 relative retention time to Ebastine peak. UV degradation product was isolated from reaction mass using preparative HPLC and its structure was elucidated using high resolution MS, multidimensional NMR and FTIR spectroscopic techniques. UV degradation product has been characterized as 2-(4-(benzhydryloxy)piperidin-1-yl)-1-(4-(tert-butyl)phenyl)-2-methylcyclopropanol. (1)H and (13)C NMR chemical shift values were generated using computational chemistry for possible two diastereomers (7R10S and 7R10R) and later 7R10R was confirmed (and its enantiomer) as final structure given it showed close agreement with experimental NMR data. Formation of UV degradation product as a recemic mixture was further verified by computational chemistry evaluation, chiral HPLC and polarimetery. To best of our knowledge, this is a novel degradation product which is not discussed at any form of publication yet.

Isolation and characterization of a novel acid degradation impurity of Amlodipine Besylate using Q-TOF, NMR, IR and single crystal X-ray.

Forced degradation of Amlodipine Besylate (AMD) in acidic condition gave rise to a potential unknown impurity. This unknown acid degradation product (ADP) was evaluated using a new-reverse-phase high performance liquid chromatography (HPLC), where it was eluted at 1.24 relative retention time to AMD peak. ADP was isolated using preparative HPLC from degradation mixture. Later, structure of ADP was elucidated using high resolution MS, multidimensional NMR and FTIR spectroscopic techniques, and characterized as ethyl-6-(2-chlorophenyl)-8-methyl-3,4,6,7-tetrahydro-2H-benzo[b][1,4]oxazine-5-carboxylate. The presence of ADP recemic mixture was confirmed by polarimeter and chiral HPLC. Given the complexity associated with ADP generation, single crystal X-ray crystallography technique was used to confirm proposed structure. In addition, reaction mechanism was postulated and confirmed using computational chemistry. To our knowledge, it is a novel impurity and not reported elsewhere.

Evaluation of degradation kinetics for abamectin in formulations using a stability indicating method.

The aim of this study was to evaluate stability characteristics and kinetics behavior of abamectin (ABM) as a 1 % (m/V) topical veterinary solution. During the study, samples stressed at 55 and 70 °C were regularly analyzed for several parameters over 8 weeks on a chromatographic (HPLC) system, using a Prodigy C18, 250 x 4.6 mm, 5-µm, column eluting with 15 : 34 : 51 (V/V/V) water/methanol/ acetonitrile as mobile phase. The HPLC method was validated for precision, accuracy, linearity and specificity, and was found to be stability indicating. The results showed that degradation of ABM followed first-order kinetics and data on loss in kobs (s-1) and half life (t1/2, days) demonstrated ABM showing the maximum stability in glycerol formal. The degradation behavior of ABM varies from solvent to solvent. The effect of added alkali on pH change and loss of ABM was studied and found to be unique for all solvents and very distinct from typical hydrolysis degradation. The present study may serve as a platform to design and develop topical non-aqueous solutions of ABM for veterinary use given no such comprehensive efforts have been published to date on the stability profile of ABM in non-aqueous solvents.

Analytical profile of moxidectin.

Moxidectin or F28249α is a potent endectocide and semisynthetic methoxime derivative of naturally occurring nemadectin. It is well known for the novel mode of action against a broad range of nematode and anthropod animal parasites. In this work, physicochemical and pharmaceutical aspects of moxidectin are described including stability, semisynthesis, purification processes, formulation compositions, impurities, and degradation pathways. Additional experiments such as DSC, XRD, and CHN analysis were carried out to complete the profile of moxidectin. The importance of safety and quality of drug substances was highlighted by chronological developments involving moxidectin and its analogues. The information gathered from the literature was used to trace the origins of moxidectin-related substances presented in the European Pharmacopeia (EP) compendial monograph. During the review, it was noticed that majority of impurities presented in the EP does not have any potential to increase with time in drug substance or formulated products; therefore, they do not require monitoring during stability studies. This also showed the requirement for further characterization of the impurities observed during long-term storage and development of stability indicating methods distinguishing between process impurities and the true degradation products. Furthermore, the stability of moxidectin in formulations is also reviewed in conjunction with known degradation routes and innovative ways to formulate products that are stable and effective at intended shelf life.

Separation and identification of degradation products in eprinomectin formulation using LC, LTQ FT-MS, H/D exchange, and NMR.

The aim of this study was to evaluate the suitability of the compendial active pharmaceutical ingredient (API) method for the analysis of finished products and characterization of degradation products in eprinomectin (EPM) samples. Heat stressed sample tests revealed a limitation of the API method in distinguishing an impurity merging with the principal analyte peak. A new selective, specific and sensitive method was therefore developed for the determination of EPM in formulations that separates its degradation products currently undetectable with the official method. The determination was carried out by reversed-phase HPLC using an isocratic solvent elution. The method was validated and found to be precise, accurate and specific; the detector response was linear over 50-150 µg/ml (EPM) and 0.1-3 µg/ml (degradation product) range of concentrations. Two major degradation products detected with the new method were isolated from sample matrices and characterized using LC-PDA, high resolution FT-ICR MS, NMR and hydrogen/deuterium exchange (HX-MS) studies. FTMS analysis showed accurate mass of molecular ion peaks for EPM and its two degradation products at m/z 914.52505 (mass error ≤ 1 ppm) with almost identical fragmentation patterns. Given the isomeric nature of the compounds, all three were further evaluated by ¹H, ¹³C, 1D NOESY and 2D (COSY) NMR experiments. The interpretation of experimental data positively identified Unknown 1 as the 2-epimer of EPM and Unknown 2 as the structural isomer Δ2,3-EPM containing a conjugated enoate. The new HPLC method and identification exercise is useful for analysis of EPM and its degradation products.