Investigation of Unusual N-(Triphenyl-λ5-phosphanylidene) Amide Fragmentation Observed upon MS/MS Collision-Induced Dissociation.

A mechanism of unusual tandem (MS/MS) fragmentation of protonated species of N-(triphenyl-λ5-phosphanylidene) derivatives, [M + H]+ to generate triphenylphosphine oxide (TPPO) within the mass spectrometer has been investigated and reported. Collision-induced dissociation of these molecules resulted in the generation of TPPO as a signature fragment. This fragment suggested the presence of a P-O bond in the structure which was contrary to the structure of the compound identified by nuclear magnetic resonance spectrometry (NMR) and single-crystal X-ray diffractometry (SXRD) techniques with a P═N bond rather than a P-O bond. In order to confirm the generation of the TPPO fragment within the mass spectrometer, 14 different N-(triphenyl-λ5-phosphanylidene) derivatives containing amide, 18O-labeled amide, thiamide, and nonacyl phosphazene derivatives were synthesized and their MS/MS behavior was studied by liquid chromatography-high-resolution mass spectrometry. Fragmentation of these amide derivatives generated TPPO/TPPS or their 18O-labeled analogues as the major fragment in almost all cases under similar MS conditions. Based on the outcome of these experiments, a plausible mechanism for such fragmentation, involving the intramolecular shifting of oxygen from carbon to phosphorus, has been proposed. DFT calculations for the protonated species at B3LYP-D3/6-31+G(d,p) further supported the proposed mechanism involving a four-membered ring, P-O-C-N, as the transition state. Details of this work are presented here.

A Tribute to Professor Saranjit Singh - A Critical Thinker, Innovator, Mentor, and Educator.

This commentary presents contributions and accomplishments of Professor Saranjit Singh, National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar, India, to pharmaceutical research and education. Prof. Singh completed his successful tenure in October 2021. Over his 40+ years of illustrious academic career, he trained 147 Masters and 15 PhD students in the fields of drug stability testing, degradation chemistry, impurity and metabolite characterization, and advanced analytical technologies. He has published ∼250 research articles, reviews, editorials, patent, book, and book chapters, and received numerous awards, including the Professor M.L. Khorana Memorial Lecture Award from the Indian Pharmaceutical Association (IPA) and the Outstanding Analyst and Eminent Analyst awards from the Indian Drug Manufacturers' Association (IDMA). This commentary highlights Prof. Singh's inspiring personal and renowned professional journey, including early life, education, career, accomplishments, as well as his services to academia, industry, and regulatory. By sharing the contributions and accomplishments of Prof. Singh, we strongly believe that his story will inspire the next generation of scientists to continue his legacy to advance the field.

Development of HPLC-CAD stability indicating assay method for polyethylene glycol-conjugated phospholipid (DMPE-PEG 2000) and identification of its degradation products.

The study introduces first report on a liquid chromatographic method for the quantification of 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] ammonium salt (DMPE-PEG 2000), which is an important constituent of lipid-based nanoparticles. It involves an HPLC-CAD stability-indicating assay method development for DMPE-PEG 2000 and structure elucidation of its degradation products. Hypersil Gold™ PFP column (150 mm × 4.6 mm, 3.0 µm) was used to achieve the separation among DMPE-PEG 2000 and its degradation products using 0.0025% formic acid in water: methanol (80:20 v/v) as mobile phase A and methanol: acetonitrile (60:40 v/v) as mobile phase B in a gradient elution mode. The method was validated for precision, linearity, sensitivity, solution stability and robustness. Relative standard deviations for the intra-day precision, inter-day precision and sensitivity were 1.6%, 0.6% and 3.8%, respectively. The method was linear in the range from 210 µg/mL to 390 µg/mL with R2 value of 0.996. Further, the solution stability of DMPE-PEG 2000 was evaluated under different stressed and storage conditions to understand the impact of any excursion to its regular storage temperature of -20 °C. The observed degradation products were identified through liquid chromatography high resolution mass spectrometry and a tentative pathway was proposed for the generation of these degradants.

Identification of extractables by liquid chromatography-high resolution mass spectrometry: A case study to understand the extraction profile of different disposable syringes.

The current study was undertaken to evaluate clinical-grade disposable syringes of various brands based on the identification of an extraneous impurity leaching out from the rubber gasket of the syringe during sample preparation for liquid chromatography method development. The syringes were evaluated using aqueous and organic solvents and their mixtures to understand susceptibility towards extractables. It was observed that the extraction propensity of different brands of syringes was varying in terms of number and levels of extractables. A total of eight extractables (including the extraneous impurity observed during method development) were identified from the syringe gaskets with the help of liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS), nuclear magnetic resonance (NMR) and/or infrared (IR) spectroscopy. To the best of authors' knowledge, four out of eight proposed extractables are not reported in the literature. A tentative pathway for the formation of the extractables was also proposed.

Rapid and efficient chiral method development for lamivudine and tenofovir disoproxil fumarate fixed dose combination using ultra-high performance supercritical fluid chromatography: A design of experiment approach.

Fixed dose combination (FDC) of tenofovir disoproxil fumarate (TDF) and lamivudine (3TC) is one of the most preferred FDC for the treatment of acquired immunodeficiency syndrome (AIDS)/human immunodeficiency virus (HIV) infection. To the best of authors' knowledge there are no reported methods for chiral purity estimation of both drugs simultaneously from a FDC. The current study was focused on the development of a single chiral method uisng supercritical fluid chromatography (SFC) for separation of stereoisomers of TDF and 3TC combination employing design of experiment (DoE) approach. Method development was planned in three steps by using different experimental designs for each step. I-optimal, Taguchi orthogonal array and face-centred central composite designs (CCD) were employed for primary parameter selection, secondary parameter screening and final method optimization, respectively. All six stereoisomers were separated in a 10 minute run on Chiralpak IA column with carbon di-oxide /methanol (containing 0.5 % v/v n-butylamine) as mobile phase at 1.5 mL/min in gradient mode. The optimized method was verified for performance through establishing specificity, precision, linearity, accuracy, limit of quantification, and solution stability. Resolution between each isomeric pair was more than 1.5. The method was found to be linear from 1.5 µg/mL to 7.5 µg/mL for 3TC and 7.5 µg/mL to 37.5 µg/mL for TDF stereoisomers. The R2 values for all the linearity curves for undesired isomers were greater than 0.995. The method proved to be rapid, reproducible and efficient to quantify stereoisomers of both drugs in a single run.

Stability behaviour of antiretroviral drugs and their combinations. 11: Characterization of interaction products of zidovudine and efavirenz, and evaluation of their anti HIV-1 activity, and physiochemical and ADMET properties.

Zidovudine (ZDV) and efavirenz (EFV), which belong to two separate classes of antiretroviral drugs, viz., NRTI and NNRTI, respectively, were subjected to different stability test conditions alone and in solid mixtures to evaluate possibility of interaction among them. The exposed samples were analyzed by high performance liquid chromatography (HPLC) using a C18 column and a PDA detector. Two new peaks were observed in the sample in which 50 µl CH3CN was added to increase the contact among the drugs, and which was subjected in open beaker to accelerated stability test condition of 40 °C/75%RH for 15 d. Subsequently, liquid chromatography-high resolution mass spectrometric (LC-HRMS) studies were carried out to obtain their accurate mass. The products were also isolated, and subjected to 1H, 13C, DEPT-135, COSY, HSQC and HMBC nuclear magnetic resonance (NMR) studies. The collective information allowed their structural characterization as isomeric cycloaddition products of the two drugs. As these were novel compounds, they were subjected to testing for cytotoxicity and in vitro anti-HIV-1 activity against primary isolates HIV-1UG070 (X4, subtype D) and HIV-1VB59 (R5, subtype C) in TZM-bl cell line. The two were found to show weak activity against the standard drugs. The reason was sought through molecular docking studies, which highlighted that it was perhaps their comparative bigger molecular size than the drugs of both classes used currently in HIV therapy. Being previously unknown molecules, their in silico physicochemical and ADMET properties were also evaluated using ADMET Predictor™ and TOPKAT software.

Stability behaviour of antiretroviral drugs and their combinations. 9: Identification of incompatible excipients.

Incompatibility studies of antiretroviral drugs, viz., lamivudine (3TC), emtricitabine (FTC), abacavir sulfate (ABC), tenofovir disoproxil fumarate (TDF), zidovudine (ZDV), efavirenz (EFV) and nevirapine (NVP) were carried out in the presence of ten selected excipients, i.e., microcrystalline cellulose, lactose monohydrate, starch, magnesium stearate, sodium lauryl sulfate, sodium starch glycolate, croscarmellose sodium, colloidal silica, povidone K-30 and hydroxypropyl cellulose. Among all, ABC showed reaction with lactose monohydrate, resulting in the formation of two interaction products, while sodium lauryl sulphate enhanced the degradation of TDF. The interaction products of ABC-Lactose were separated by high performance liquid chromatography (HPLC) and subjected to liquid chromatography-high resolution mass spectrometry (LC-HRMS) studies for their characterization. One of the products was also isolated and subjected to 1D and 2D nuclear magnetic resonance (NMR) studies for structural confirmation. The toxicity of both was predicted using TOPKAT and ADMET™ software and compared to the drug.

Characterization of solution stress degradation products of aliskiren and prediction of their physicochemical and ADMET properties.

Forced degradation studies on aliskiren were carried out according to ICH and WHO guidelines. Six degradation products were formed in total in the solution state. Their separation among themselves and from the drug was successfully achieved on a C-18 column utilizing acetonitrile and phosphate buffer (pH 3.0) in the mobile phase, which was run in a gradient mode. To characterize them, a complete mass fragmentation pathway of the drug was first established with the help of MS/TOF and MSn data. This was followed by LC-MS/TOF studies on the degradation products. Some of the degradation products were also isolated and subjected to 1D (1H, 13C and DEPT-135) and 2D (COSY, HSQC and HMBC) NMR studies for confirmation of their structures. An interesting observation was hydrolysis followed by cyclization in case of three degradation products. Also, acetonitrile was found to react with aliskiren, leading to formation of a pseudo degradation product. Additionally, comparative ADMET properties of the drug and degradation products were established using ADMET Predictor™.

Successful Development by Design of Experiments of a Gas Chromatography Method for Simultaneous Analysis of Residual Solvents of Classes 1 and 2.

In this study, design of experiments (DoE) was employed to develop a single injection method using a headspace gas chromatograph with flame ionization detector for resolution of residual solvents of United States Pharmacopeia (USP) listed classes 1 and 2, against current recommendation of independent injections. G43 column (6% cyanopropylphenyl and 94% dimethylpolysiloxane) and nitrogen were used as the stationary phase and carrier gas, respectively. Initial temperature, hold time, temperature ramp and carrier gas velocity were the critical method parameters. Resolution of 1,1,1-trichloroethane and tetrahydrofuan (THF); THF and chloroform; benzene and 1,2-dichloroethane (DCE), and 1,2-dimethoxyethane and DCE were selected as critical quality attributes. These were optimized by DoE that resulted in resolution of >1.34 among various solvents. The separation of all the solvents was achieved within total run time of 77 min. A better resolution of 2.66 was observed in the case of acetonitrile and methylene chloride; there was improved Signal/Noise ratio of 8.86 for 1,1,1-trichloroethane; tailing factor for pyridine was 1.00, and the method showed acceptable repeatability of peak areas (%RSDmax = 11.53) and retention times (%RSDmax = 0.45). Thus, while system suitability criteria and validation results very well met the USP requirements, the optimized method proposed in this study proved advantageous additionally in terms of single injection; short run time, and use of nitrogen as a carrier gas instead of costly helium in the USP method.

Stability behaviour of antiretroviral drugs and their combinations. 8: Characterization and in-silico toxicity prediction of degradation products of efavirenz.

Efavirenz (EFV), an antiretroviral drug, was evaluated for its degradation behaviour in solution state. A total of twelve degradation products were detected on high performance liquid chromatography (HPLC) analyses. Initially, comprehensive mass fragmentation pattern of the drug was established by direct injection and collection of high resolution mass spectrometry (HRMS) and multi-stage tandem mass spectrometry (MSn) data. Subsequently, LC-HRMS studies were carried on the stability samples containing the degradation products. Eleven degradation products were isolated and subjected to 1D and 2D nuclear magnetic resonance (NMR) studies for their structural confirmation. The collated information was utilized for the characterization of all the degradation products and hence in outlining the comprehensive degradation pathway of the drug. In-silico toxicity of the degradation products was evaluated by TOPKAT analyses.

Characterization of forced degradation products of torasemide through MS tools and explanation of unusual losses observed during mass fragmentation of drug and degradation products through density functional theory.

Mass spectrometry tools (HRMS/LC-HRMS, MSn, and/or on-line H/D exchange) were employed to establish mass fragmentation pattern of torasemide and to characterize its degradation products. During collision-induced dissociation, multiple rearrangement processes and unusual losses of sulfur (S), sulfanyl (HS), sulfur dioxide (SO2), sulphinic acid radical (HSO2), sulfur monoxide (SO), carbon monoxide (CO), formyl radical (CHO) and C5H3NOS were observed. The same were successfully explained by study of energy profiles, established by application of density functional theory (DFT).

Stability behavior of antiretroviral drugs and their combinations. 7: Comparative degradation pathways of lamivudine and emtricitabine and explanation to their differential degradation behavior by density functional theory.

The interest in this study was to establish comparative degradation behavior of lamivudine (3TC) and emtricitabine (FTC) under solution and solid state stress conditions. Structurally, the two drugs differ only in terms of additional fluorine at 5 position in FTC. Along with the known degradation products of both the drugs, one additional degradation product was observed in each case, which was characterized by mass spectrometry. Both the drugs degraded via the same route, but at a differential rate in acid, base and oxidative stress conditions. The variable rate of degradation in acid and base conditions was justified by the application of density functional theory (DFT).

Stability behaviour of antiretroviral drugs and their combinations. 5: Characterization of novel degradation products of abacavir sulfate by mass and nuclear magnetic resonance spectrometry.

In the present study, degradation behaviour of abacavir sulfate was evaluated in solution and solid stress conditions. Solution state studies resulted in formation of eleven degradation products; of which two were also formed on solid stress. The same were separated by high performance liquid chromatography. They were characterized using liquid chromatography-high resolution mass spectrometry, liquid chromatography-multistage mass spectrometry and hydrogen/deuterium exchange mass spectrometry data. Additionally, seven degradation products were isolated and subjected to 1D and 2D nuclear magnetic resonance studies for their structural confirmation.

Stability behaviour of antiretroviral drugs and their combinations. 4: Characterization of degradation products of tenofovir alafenamide fumarate and comparison of its degradation and stability behaviour with tenofovir disoproxil fumarate.

In this study, stress degradation behaviour of tenofovir alafenamide fumarate (TAF), a novel prodrug of tenofovir, was investigated and compared with currently used prodrug congener, tenofovir disoproxil fumarate (TDF), whose intrinsic stability was reported by us earlier [14]. Also, pH stability and gastrointestinal stability studies were conducted on both the drugs. High performance liquid chromatography (HPLC) analysis of stressed samples of TAF revealed formation of six degradation products (DPs) against twelve characterized earlier in the case of TDF (RSC Adv. 5(2015) 96117-96129). Like TDF, characterization of DPs of TAF was done by using sophisticated hyphenated liquid chromatography-high resolution mass spectrometry (LC-HRMS) and multistage mass spectrometry (MSn) tools. pH-stability studies between pH 1.2-10 revealed greater stability of TAF, except in acidic conditions, where TAF was degraded extensively. Investigation of gastrointestinal stability in simulated gastric fluid (SGF), simulated intestinal fluid (SIF) and fed state simulated gastric fluid (FeSSGF) suggested that TAF must be administered in fed state, as the drug was practically stable in FeSSGF as compared to extensive loss at acidic pH and in SGF.

Stability behaviour of antiretroviral drugs and their combinations. 3: Characterization of interaction products of emtricitabine and tenofovir disoproxil fumarate by mass spectrometry.

The present study investigated drug-drug interaction behaviour of emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) under solid state stability test conditions. Six interaction products were separated and detected by high performance liquid chromatography coupled to photodiode array detector (HPLC-PDA) using C18 column. The same were characterized using LC-high resolution mass spectrometry (LC-HRMS), LC-multi stage mass spectrometry (LC-MS(n)) and online hydrogen/deuterium (H/D) exchange studies. The interaction pathway among the two drugs was outlined based on the elucidated structures. Four of the six interaction products were also formed in marketed tablets containing FTC and TDF (along with efavirenz (EFV)) that were kept without packing under accelerated condition of 40°C/75% RH till 6 months.

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.

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 D2 O 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 (r(2) = 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 © 2016 John Wiley & Sons, Ltd.

Implementation of design of experiments for optimization of forced degradation conditions and development of a stability-indicating method for furosemide.

The study involved optimization of forced degradation conditions and development of a stability-indicating method (SIM) for furosemide employing the design of experiment (DoE) concept. The optimization of forced degradation conditions, especially hydrolytic and oxidative, was done by application of 2(n) full factorial designs, which helped to obtain the targeted 20-30% drug degradation and also enriched levels of degradation products (DPs). For the selective separation of the drug and its DPs for the development of SIM, DoE was applied in three different stages, i.e., primary parameter selection, secondary parameter screening and method optimization. For these three, IV-optimal, Taguchi orthogonal array and face-centred central composite designs were employed, respectively. The organic modifier, buffer pH, gradient time and initial hold time were selected as primary parameters. Initial and final organic modifier percentage, and flow rate came out as critical parameters during secondary parameter screening, which were further evaluated during method optimization. Based on DoE results, an optimized method was obtained wherein a total of twelve DPs were separated successfully. The study also exposed the degradation behaviour of the drug in different forced degradation conditions.