Mallikarjun Narayanam

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.

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.

A systematic strategy for the identification and determination of pharmaceuticals in environment using advanced LC-MS tools: application to ground water samples.

In the present study, a novel analytical strategy was employed to study the occurrence of 40 drug residues belonging to different medicinal classes, e.g., antibiotics, β blockers, NSAIDs, antidiabetics, proton pump inhibitors, H2 receptor antagonists, antihypertensives, antihyperlipidemics, etc. in ground water samples collected from villages adjoining to S.A.S. Nagar, Punjab, India. The drug residues were extracted from the samples using solid-phase extraction, and LC-ESI-HRMS and LC-ESI-MS/MS were used for identification and quantitation of the analytes. Initially, qualifier and quantifier MRM transitions were classified for 40 targeted drugs, followed by development of LC-MS methods for the separation of all the drugs, which were divided into three categories to curtail overlapping of peaks. Overall identification was done through matching of retention times and MRM transitions; matching of intensity ratio of qualifier to quantifier transitions; comparison of base peak MS/MS profiles; and evaluation of isotopic abundances (wherever applicable). Final confirmation was carried out through comparison of accurate masses obtained from HRMS studies for both standard and targeted analytes in the samples. The application of the strategy allowed removal of false positives and helped in identification and quantitation of diclofenac in the ground water samples of four villages, and pitavastatin in a sample of one village.

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.

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.

Characterization of stress degradation products of fosinopril by using LC-MS/TOF, MSn and on-line H/D exchange

Fosinopril was subjected to stress degradation studies under hydrolytic (acidic, basic and neutral), oxidative, photolytic and thermal stress conditions. The drug underwent degradation in hydrolytic and photo acidic conditions, while it was stable to oxidative and thermal stress. The degradation products (DPs) were detected after using a gradient LC-MS method. These were characterized using a protocol involving comparison of mass fragmentation pathways of the drug and the DPs, and consideration of mass data generated during MS/TOF, MS(n) and H/D exchange studies. The degradation pathway of the drug was established, which was duly justified by mechanistic explanation.

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

Validated Stability-Indicating Method for Alendronate Sodium Employing Zwitterionic Hydrophilic Interaction Chromatography Coupled with Charged Aerosol Detection

Alendronate sodium is widely used in the treatment of osteoporosis and Paget’s disease. The HPLC method development for alendronate sodium, in particular, is challenging owing to the absence of chromophoric group and its high polarity. In the present study, a short and simple isocratic method was developed involving hydrophilic interaction liquid chromatography, coupled with a charged aerosol detector. The developed method was validated according to the ICH Q2(R1) guideline and was successfully applied for the analysis of a marketed formulation containing the drug.