Forced degradation study of racecadotril: Effect of co-solvent, characterization of degradation products by UHPLC-Q-TOF-MS/MS, NMR and cytotoxicity assay.

Racecadotril, an enkephalinase inhibitor, was subjected to hydrolysis (acidic and alkaline), oxidation, photolysis and thermal stress, as per ICH specified conditions. The drug showed extensive degradation under acidic, basic hydrolysis and oxidative stress conditions whereas, it was stable under other stress conditions. A total of seven degradation products (DPs) were observed. The chromatographic separation was optimized on Acquity HSS Cyano (100×2.1mm, 1.8μ) column using 0.1% formic acid and acetonitrile as mobile phase in gradient mode. Six DPs were characterised by LC-MS/MS and DP1 by GC-MS. The major DPs (DP 2 and DP 5) were isolated and characterised by NMR. This is a typical case of degradation where co solvent methanol reacts with racecadotril leading to the formation of pseudo DPs, DP 6 and DP 5. Interestingly the MS/MS spectra of protonated drug, DP 4 and DP 7 showed product ions which were formed due to intramolecular benzyl migrations. In vitro cytotoxic activity studies on isolated DP 2 and DP 5 revealed that the former has no cytotoxic nature, whereas the latter has potential pulmonary and hepatic toxicity.

Chiguru V ，Lingesh A ，R S ，N S ... -  《-》

Forced degradation of fingolimod: effect of co-solvent and characterization of degradation products by UHPLC-Q-TOF-MS/MS and 1H NMR.

Fingolimod (FGL), an immunomodulator drug for treating multiple sclerosis, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per International Conference on Harmonization specified conditions. The drug showed extensive degradation under base hydrolysis, however, it was stable under all other conditions. A total of three degradation products (DPs) were observed. The chromatographic separation of the drug and its degradation products was achieved on a Fortis C18 (100×2.1mm, 1.7μm) column with a mobile phase composed of 0.1% formic acid (Solvent A) and acetonitrile (Solvent B) in gradient mode. All the DPs were identified and characterized by liquid chromatography-quadrupole time of flight-mass spectrometry (LC-Q-TOF-MS) in combination with accurate mass measurements. The major DP was isolated and characterized by Nuclear Magnetic resonance spectroscopy. This is a typical case of degradation where acetonitrile used as co-solvent in stress studies, reacts with FGL in base hydrolytic conditions to produce acetylated DPs. Hence, it can be suggested that acetonitrile is not preferable as a co-solvent for stress degradation of FGL. The developed UHPLC method was validated as per ICH guidelines.

Patel PN ，Kalariya PD ，Gananadhamu S ，Srinivas R ... -  《-》

Identification and characterization of forced degradation products of sertraline hydrochloride using UPLC, ultra-high-performance liquid Chromatography-Quadrupole-Time of flight mass spectrometry (UHPLC-Q-TOF/MS/MS) and NMR.

The present study focused on the forced degradation behavior of sertraline hydrochloride (SRT), selective serotonin reuptake inhibitor (SSRI). The drug was exposed to different stressed conditions (hydrolytic, oxidative, thermal and photolytic) according to ICH Q1A (R2) guidelines. The study revealed that SRT was stable in hydrolytic (acidic, basic and neutral) and thermal degradation conditions. In contrast, five degradation products (DPs) were formed under oxidative and photolytic degradation conditions. The chromatographic separation of drug substance and its DPs was achieved on an Acquity HSS T3 column (100 × 2.1 mm, 1.7 μ) using 0.1% formic acid and acetonitrile as the mobile phase in gradient mode using a UHPLC-DAD system. The DPs were identified and characterized by high-resolution LC/MS and LC/MS/MS in ESI positive mode. Two DPs (DP-I and DP-II) were formed when SRT was exposed to oxidative degradation conditions. Three DPs formed (DP III-V) when exposed to photolytic degradation conditions. All the five major DPs were isolated using Preparative HPLC. The structures of major DPs formed were further confirmed using NMR technique (1D and 2D). The proposed mechanism for the formation of the SRT DPs via the photolytic/oxidative stress degradation pathway are discussed and outlined.

Grover P ，Bhardwaj M ，Mukherjee D 《-》

Isolation, characterization using LC-ESI-QTOF, NMR and in vitro cytotoxicity assay of niclosamide forced degradation products.

The present study describes the isolation, characterization and in vitro cytotoxic effect of all forced degradation products of niclosamide (NCM) an anthelmintic class of drug used specifically to treat tapeworms. NCM was subjected to forced degradation involving hydrolysis (acidic, alkaline and neutral), oxidative, photolysis and thermal stress, as per ICH (Q1A (R2)) suggested conditions. The drug under hydrolytic (acidic and basic) conditions showed extensive degradation, while it was stable under neutral hydrolytic, oxidative, photolytic and thermal stress conditions. A total of four degradation products (DPs) were observed and chromatographic separation of drug and its degradation products were achieved on a reverse phase Fortis diphenyl column (150×4.6mm, 5μm) using 0.1% formic acid and acetonitrile as mobile phase in gradient mode. All the four degradation products were isolated by semi preparative LC and its structures were characterized and confirmed by high resolution MS and 1H NMR spectroscopic techniques. In view of safety aspects, cytotoxicity assay were carried out for NCM and its four degradation products on human mononuclear cells and cell lines depicting the major organelle: neuronal (Neuro 2a), hepatic (HepG 2) and alveolar (A549). NCM was found to be non toxic on human mononuclear cells and cell lines at tested concentrations. However DP-1, DP-2, DP-3 and DP-4 showed significant increase in LDH release as compared to control at a concentration of 100μM. DP-1 and DP-3 exhibited toxicity on A549 cells with an IC50 of 92.18±4.93μM and 65.42±6.29μM respectively. DP-2, DP-3 and DP-4 were cytotoxic to Neuro 2a cells with an IC50 of 63.62±3.85μM, 86.09±6.19μM and 42.81±8.10μM respectively. The degradation products were found to be nontoxic on HepG 2 cells.

P J ，Ch V ，A L ，V G M N ，Ch N ，N S ... -  《-》

Validated stability indicating assay method of olaparib: LC-ESI-Q-TOF-MS/MS and NMR studies for characterization of its new hydrolytic and oxidative forced degradation products.

Olaparib (OLA) is a poly ADP ribose polymerase (PARP) enzyme inhibitor used to treat prostate, ovarian and breast cancer. The drug substance OLA was subjected to forced degradation as per ICH prescribed guidelines. It was degraded in hydrolytic (acidic and basic) and oxidative stress conditions and yielded four degradation products (DPs) while it remained stable in neutral hydrolytic, dry heat and photolytic stress conditions. A stability indicating assay method was developed to separate OLA and its DPs using InertSustain C18 column (250 × 4.6 mm, 5 μm) with a gradient mobile phase of 10 mM ammonium acetate (pH 4.5) and acetonitrile (ACN) at a flow rate of 1 mL min-1. The characterization of DPs was carried out by using liquid chromatography-electrospray ionization-quadrupole-time of flight tandem mass spectrometry (LC-ESI-Q-TOF-MS/MS). Major degradation products (DP-1 and DP-2) were isolated by using preparative HPLC and structures were further confirmed by using NMR spectroscopy. All the obtained DPs were new and not reported previously. The developed chromatographic method was validated as per ICH Q2 (R1) guideline and USP general chapter on method validation.

Thummar M ，Kuswah BS ，Samanthula G ，Bulbake U ，Gour J ，Khan W ... -  《-》