Development of stability-indicating method for separation and characterization of benidipine forced degradation products using LC-MS/MS.

The present study describes forced degradation of benidipine (BEN) as per  Q1A (R2) and Q1B guidelines of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. BEN degraded under hydrolysis (neutral, acidic, and alkaline), hydrogen peroxide induced oxidation, and UV light mediated photolytic degradation. A total of 14 degradation products (DPs) were found in all degradation studies, comprising 4 hydrolytic DPs, 8 oxidative DPs, and 4 photolytic DPs. A selective stability-indicating method was developed using an XBridge BEH C18 column with gradient elution program consisting of ammonium acetate (10 mM, 4.8 pH, acetic acid) and acetonitrile. The flow rate was maintained at 1 ml min-1 . All DPs were separated well using the developed HPLC method and were characterized using LC-MS/MS data. As this method is effective in identifying and separating BEN and its DPs with sufficient resolution, it can be used in laboratories for quality control of drugs in daily routine analysis and stability studies.

Characterization of potential degradation products of brexpiprazole by liquid chromatography/quadrupole-time-of-flight mass spectrometry and nuclear magnetic resonance, and prediction of their physicochemical properties by ADMET Predictor™.

RATIONALE: Brexpiprazole (BRZ) was subjected to hydrolytic (acid, base and neutral), oxidative, photolytic and thermal stress degradation in solutions prepared in a mixture of acetonitrile-water (70:30 v/v). The oxidative study was additionally done in methanol-buffer mixture at pH 3, 7 and 11. Also, compatibility of the drug with selected excipients was investigated in the solid state. Additionally, physicochemical and ADMET properties of BRZ and its degradation products (DPs) were predicted using ADMET Predictor™ software. It provides the conditions for quality control of BRZ and its derivatives during manufacturing, processing and storage conditions. METHODS: The formed DPs were separated from the drug and among themselves on a C-18 column utilizing mobile phase composed of methanol and ammonium formate buffer (10mM, pH 4.0), which was run in a gradient mode. Characterization of DPs was carried out by first establishing the mass fragmentation pathway of the drug based on its liquid chromatography/quadrupole-time-of-flight mass spectrometry (LC/Q-TOF-MS) data, followed by LC/Q-TOF-MS studies of DPs. Three DPs were isolated and, along with the drug, they were subjected to 1D (1 H, 13 C and DEPT-135) and 2D (COSY and HSQC) NMR studies for confirmation of their structures. RESULTS: BRZ was observed to be susceptible to hydrolytic (neutral, acid and alkali), photolytic and oxidative degradation conditions; it was stable on thermal exposure. A total of 12 DPs (BRZ-1 to BRZ-12) were formed in solution state. Mechanisms of BRZ degradation were postulated. CONCLUSIONS: The extent of degradation of BRZ in different stress conditions highlights that stability of BRZ in drug formulations can be improved (i) by using excipients that can impart a low-pH microenvironment, (ii) by addition of antioxidants and (iii) through protection from light.

Preparation and Comparison of Oral Bioavailability for Different Nano-formulations of Olaparib.

Olaparib (OLA) is a poly ADP ribose polymerase (PARP) inhibitor approved for germline BRCA-mutated (gBRCAm) advanced ovarian cancer and breast cancer. Low oral bioavailability of this drug requires increase in the dose and frequency causing haematological toxicity in the patients. The purpose of this study is to prepare different nano-formulations of OLA lipospheres (LP) by melt dispersion and nano-suspensions (NSP) by solvent evaporation (SE) and wet milling (WM) techniques and compare oral bioavailability of these formulations. Size of the nano-formulations OLA-LP, OLA-NSPSE and OLA-NSPWM were found to be 126.71 ± 4.54, 128.6 ± 2.34 and 531.1 ± 5.34 nm with polydispersity index below 0.3. In vitro release studies were performed by dialysis bag method where the sustained drug release was observed from nano-formulations until 9 h with Higuchi for OLA suspended in 2.5% w/v sodium carboxy methyl cellulose (OLA-SP), OLA-LP and OLA-NSPWM and Peppas for OLA-NSPSE-based drug release kinetics. In vivo pharmacokinetic studies, haematological toxicity and distribution studies were performed on rats. Results showed that there was an improvement in Cmax, AUCtotal, t1/2 and MRT by OLA nano-formulations when compared with OLA-SP. OLA-SP has shown reduction in WBC, platelets and lymphocytes at 12 and 36 h time points; however, no reduction in cell count was observed with OLA nano-formulations. Distribution studies proved FITC nano-formulations were most rapidly absorbed and distributed when compared with FITC-loaded suspension. From the above results, it was concluded that OLA nano-formulations can be an alternative to enhance the oral bioavailability and to reduce the haematological toxicity of OLA.

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.

Stability-indicating assay method for acotiamide: Separation, identification and characterization of its hydroxylated and hydrolytic degradation products along with a process-related impurity by ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

RATIONALE: The presence of impurities and degradation products will affect the pharmacokinetic, pharmacodynamic properties and alter the safety of a drug. Hence, the development of a stability-indicating assay method is an integral part of quality product development and is crucial for the regulatory approval of drug products. METHODS: Acotiamide was subjected to stress degradation under hydrolytic, oxidative, photo and thermal stress conditions. The resulted degradation products (DPs), as well as a process-related impurity (IMP), were selectively separated from the drug on a Waters Acquity HSS cyano column (100 × 2.1 mm, 1.8 µm) with a mobile phase containing a gradient mixture of 0.1% formic acid and acetonitrile (ACN) at a flow rate of 0.25 mL min-1 . RESULTS: The drug was found to degrade under hydrolytic (acidic and basic), oxidative and photolytic stress while it remained stable under neutral hydrolytic and thermal stress conditions. The seven degradation products (DPs) and one process-related impurity (IMP) were observed. All the DPs and process-related IMP were well separated by the developed ultra-high-performance liquid chromatography (UHPLC) method and subsequently characterized by UHPLC/electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS). The proposed UHPLC method was validated with respect to specificity, linearity, accuracy, precision and robustness as per ICH guideline, Q2 (R1). CONCLUSIONS: All the observed DPs were new and formed by hydrolysis of an amide bond, phenyl ring hydroxylation and hydrolysis of the methoxy group of the phenyl ring. The despropyl process-related impurity was observed and well separated from the drug. The proposed UHPLC mass spectrometric method has greater utility in the identification of DPs in much less time with excellent selectivity.

Identification of degradation products of saquinavir mesylate by ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry and its application to quality control.

RATIONALE: Saquinavir mesylate (SQM) is an antiviral drug used for the treatment of HIV infections. The identification and characterization of all degradation products are essential for achieving the quality in pharmaceutical product development and also for patient safety. METHODS: The drug was subjected to hydrolytic (HCl, NaOH and water), oxidative (H2 O2 ), photolytic (UV and fluorescence light) and thermal (dry heat) forced degradation conditions as per ICH guidelines. The best chromatographic separation of the drug and all degradation products (DPs) was achieved on a CSH-Phenyl Hexyl column (100 × 2.1 mm, 1.7 µm) with ammonium acetate (10 mM, pH 5.0) and methanol as mobile phase in gradient mode at a flow rate of 0.28 mL/min. RESULTS: Nine DPs were obtained under various forced degradation conditions. All the DPs were characterized by using ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC/ESI-QTOF MS/MS) and the degradation pathway of the drug was justified by mechanistic explanations. The main DPs were formed by amide hydrolysis, conversion into diastereomers, an N-oxide and dehydration as well as oxidation of the alcohol from the drug. The method was validated and can be used in a quality control (QC) laboratory to assure the quality of SQM in bulk and finished formulations. CONCLUSIONS: A simple UHPLC/photodiode array (PDA) method was developed and successfully transferred to UHPLC/ESI-Q-TOF MS/MS for the identification and characterization of DPs. Very interestingly, diastereomeric DPs were obtained and successfully resolved by the chromatographic method. Copyright © 2017 John Wiley & Sons, Ltd.

In vivo metabolite identification of acotiamide in rats using ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry.

Acotiamide hydrochloride (ACT) is a drug used for the treatment of functional dyspepsia. Understanding which metabolites are likely to be formed in vivo is essential for interpreting pharmacology, pharmacokinetic and toxicology data. The metabolism of ACT has been investigated using a specific and sensitive liquid chromatography positive ion electrospray ionization high-resolution tandem mass spectrometry method. In vivo samples including rat plasma, urine and feces were collected separately after dosing healthy Sprague-Dawley rats at a dose of 20 mg kg -1 ACT at different time points up to 24 h. The metabolites were enriched by optimized sample preparation involving protein precipitation using acetonitrile followed by solid-phase extraction. The mass defect filter technique was used for better detection of both predicted and unexpected drug metabolites with the majority of interference ions removed. The structural elucidation of the metabolites was performed by comparing their [M + H]+ ions and their product ions with those of the parent drug. As a result, a total of seven hitherto unknown metabolites were characterized from the biosamples. The only phase I metabolite detected was N-despropyl acotiamide, whereas six phase II glucuronide conjugate metabolites were identified.