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This article described the development and validation of a method for spiramycin related substances based on hybrid particle column. The chromatographic conditions were as follows: water - 0.2 mol/L dipotassium hydrogen phosphate (the pH value adjusted to 9.5 using a 1 mol/L KOH solution) - acetonitrile - methanol (10: 60: 28.5: 1.5, v/v/v/v) as mobile phase A, water - 0.2 mol/L dipotassium hydrogen phosphate (pH 9.5) - acetonitrile - methanol (10: 30: 57: 3, v/v/v/v) as mobile phase B and gradient elution was performed. Compared with previous analytical methods, this method has strong specificity, excellent sensitivity and stability, which could be used for the daily testing of related substances of spiramycin. Furthermore, impurities above 0.1 % were characterized using two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometer (2D LC-QTOF-MS/MS) and there were 6 impurities reported for the first time., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Omeprazole (OME) is a proton pump inhibitor used to treat gastroesophageal reflux disease associated conditions. The current study presents an Analytical Quality by Design-based approach for the development of a CE method for OME impurity profiling. The scouting experiments suggested the selection of solvent modified Micellar ElectroKinetic Chromatography operative mode using a pseudostationary phase composed of sodium dodecyl sulfate (SDS) micelles and n-butanol as organic modifier in borate buffer. A symmetric three-level screening matrix 3 7 //16 was used to evaluate the effect of Critical Method Parameters, including Background Electrolyte composition and instrumental settings, on Critical Method Attributes (critical resolution values, OME peak width and analysis time). The analytical procedure was optimized using Response Surface Methodology through a Central Composite Orthogonal Design. Risk of failure maps made it possible to define the Method Operable Design Region, within which the following optimized conditions were selected: 72 mM borate buffer pH 10.0, 96 mM SDS, 1.45 %v/v n-butanol, capillary temperature 21 °C, applied voltage 25 kV. The method was validated according to ICH guidelines and robustness was evaluated using a Plackett-Burman design. The developed procedure enables the simultaneous determination of OME and seven related impurities, and has been successfully applied to the analysis of pharmaceutical formulations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Potential genotoxic impurities in medications are an increasing concern in the pharmaceutical industry and regulatory bodies because of the risk of human carcinogenesis. To prevent the emergence of these impurities, it is crucial to carefully examine not only the final product but also the intermediates and key starting material (KSM) used in drug synthesis. During the related substances analysis of KSM of Famotidine, an unknown impurity in the range of 0.5-1.0% was found prompting the need for isolation and characterization due to the possibility of its to infiltrate into the final product. In this study, the impurity was isolated and characterized as 5-(2-chloroethyl)-3,3-dimethyl-3,4-dihydro-2H-1,2,4,6-thiatriazine 1,1-dioxide using multiple instrumental analysis, uncovering a structural alert that raises concern. Considering the potential impact of impurity on human health, an in silico genotoxicity assessment was established using Derek and Sarah tool in accordance with ICH M7 guideline. Furthermore, molecular docking and molecular dynamics simulation were performed to evaluate the specific interaction of the impurity with DNA. The findings reveal consistent interaction of the impurity with the dG-rich region of the DNA duplex and binding at the minor groove. Both in silico prediction and molecular dynamic study confirmed the genotoxic character of the impurity. The newly discovered impurity in famotidine has not been reported previously, and there is currently no analytical method available for its identification and control. A highly sensitive HPLC-UV method was developed and validated in accordance with ICH requirements, enabling quantification of the impurity at trace level in famotidine ensuring its safe release.

Technical advances in the field of quality analysis allow an increasingly deeper look into the impurity profile of drugs. The ability to detect unexpected impurities in addition to known impurities ensures the supply of high-quality drugs and can prevent recalls due to the detection of harmful unexpected impurities, as has happened recently with the N-nitrosamine and azido impurities in losartan (LOS) drug products. In the present study, the LC-MS/HRMS approach described by Backer et al. was applied to an even more complex system, being the investigation of 35 LOS drug products and combination preparations purchased in 2018 and 2022 in German pharmacies. The film-coated tablets were analysed by means of four LC-MS/HRMS method variants. For the separation a Zorbax RR StableBond C18 column (3.0 ×100 mm, particle size of 3.5 µm, pore size of 80 Å), a gradient elution and for mass spectrometric detection a qTOF mass spectrometer with electrospray ionization in positive and negative mode was used. An information-dependent acquisition method was applied for the acquisition of high-resolution mass spectrometry data. The combination of an untargeted and a targeted screening approach revealed the finding of eight impurities in total. Beside the five LOS related compounds, LOS impurity F, J, K, L, M, and related compound D from amlodipine besilate, LOS azide and an unknown derivative thereof were detected. Identification and structure elucidation, respectively, were successfully performed using in silico fragmentation. Differences in the impurity profiles of drug products from 2018 and 2022 could be observed. This study shows that broad screening approaches like this are applicable to the analysis of drug products and can be an important enhancement of the quality assurance of medicinal products., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Laura Backer reports financial support was provided by Frankfurt Foundation Quality of Medicines. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Ganirelix, a peptide-based drug used to treat female infertility, has been in high market demand, which attracted generic formulation. A hitherto unknown impurity of ganirelix was observed in our formulation process, which reached ~0.3% in 6 months and led to a detailed investigation of its structure. In-depth analysis of ESI-MS/MS data of this impurity coupled with an artificial intelligence prediction tool led to a highly unusual putative structure, that is, N-(2-carboxyethyl)-ganirelix ( N CE-GA), which was authenticated by chemical synthesis from ganirelix and NMR analysis and via corroborated HPLC and MS/MS data with the formulation-derived impurity., (© 2023 European Peptide Society and John Wiley & Sons Ltd.)

A generic performance comparison strategy has been developed to evaluate the impact of mobile-phase additives (ion-pairing agent / counter ion systems), distinct stationary phases on resulting resolving power, and MS detectability of oligonucleotides and their critical impurities in gradient IP-RPLC. Stationary-phase considerations included particle type (core-shell vs. fully porous particles), particle diameter, and pore size. Separations were carried out at 60°C to optimize mass transfer (C-term). The incorporation of an active column preheater mitigated thermal mismatches, leading to narrower peaks and overcoming peak splitting. Acetonitrile as organic modifier outweighed methanol in terms of peak-capacity generation and yielded a 30% lower back pressure. Performance screening experiments were conducted varying ion-pairing agents and counter ions, while adjusting gradient span achieved an equivalent effective retention window. Hexafluoromethylisopropanol yielded superior chromatographic resolution, whereas hexafluoroisopropanol yielded significantly higher MS detection sensitivity. The 1.7 µm core-shell particle columns with 100 Å pores provided maximum resolving power for small (15-35 mers) oligonucleotides. Sub-min analysis for 15-35 polyT ladders was achieved operating a 50 mm long column at the kinetic performance limits. High-resolution separations between a 21-mer modified RNA sequence oligonucleotides and its related (shortmer and phosphodiester) impurities and complementary strand were obtained using a coupled column set-up with a total length of 450 mm., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aclidinium bromide (ACL) is a long-acting muscarinic receptor antagonist used for the long-term treatment of chronic obstructive pulmonary disease (COPD). The aim of this study was to investigate the degradation of aclidinium bromide under stress and stability testing conditions, for which we developed and validated the first stability-indicating, specific, precise, accurate, and robust assay and related substances HPLC methods. Nine of the compounds used as reference standards were synthesized and fully characterized by 1 H and 13 C NMR, MS, and FTIR techniques. Two of these molecules, namely ACL-dimer and ACL-desphenyl, are novel compounds and reported herein for the first time. Hydrolysis of aclidinium resulted in major degradation via the formation of ACL-desDTG and DTGA metabolites. ACL-desphenyl and phenol were observed only under oxidative conditions at very low levels (< 0.10%), while ACL-hydroxy, known as a metabolite of aclidinium and confirmed by LC/QDa and LC/Q-TOF m/z data, formed under oxidative stress-testing conditions, UV light, and daylight. The identification of two impurities formed only when aclidinium bromide was treated with hydrogen peroxide was done by LC/QDa and LC/Q-TOF studies and the novel structures were proposed as ACL-bromophenoxy and ACL-bromothiophenyl formed via bromination of the phenyl and thiophene ring on the aclidinium, respectively. The stress and photostability testing studies showed that the aclidinium bromide drug substance is not sensitive to elevated temperature (105 °C, 10 days), slightly sensitive to daylight and UV-radiation, and it showed significant degradation under all hydrolysis and oxidizing conditions. The related substances HPLC method reported herein is also capable to monitor the potential genotoxic impurity 3-bromopropoxybenzene (3-BPB), a class 2 impurity according to ICH M7, and ensures that the impurity remains below the threshold of toxicological concern (TTC) limit, making it safe for patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Oligonucleotides constitute an emerging and highly complex bioanalytical challenge and it is becoming increasingly clear that 1D methodologies are unable to fully resolve all possible impurities present in these samples. 2D-LC therefore constitutes a perfect solution wherein critical pairs can be sampled from a steep gradient 1 D and separated in a shallower 2D gradient. Herein, we provide a facile 2D-LC method development approach to quickly generate high selectivity gradients utilizing ion pairing reverse phase (IPRP-IPRP). In particular we demonstrate how to iteratively generate a 12 % gradient from two training runs and then to utilize that data to predict retentions of analytes with a 2 % gradient with retention prediction errors as low as 3 and 11 %, respectively. This iterative method development workflow was applied to impurity profiling down to 1:1000 for the full-length product and phosphorothioate modified impurities. Additionally, we demonstrated the elucidation of critical pairs in complex crude pharmaceutical oligonucleotide samples by applying tailored high selectivity gradients in the second dimension. It was found that the iterative retention modeling approach allows fast and facile 2D-LC method development for complex oligonucleotide separations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

The organic impurity profile of 3,4-methylenedioxyamphetamine (MDA) synthesised from helional via the "twodogs" method was examined to identify route-specific and condition-specific impurities. The synthesis used a condensation reaction, followed by a Beckmann rearrangement, then Hofmann rearrangement, and then conversion to a hydrochloride salt. Two chlorinating agents were investigated for the Hofmann rearrangement reaction, trichloroisocyanuric acid (TCCA) and sodium hypochlorite. Three route-specific impurities were identified in MDA using TCCA, and two of these impurities were condition-specific such that the impurities that formed were dependent on the alcohol used as solvent. Three additional impurities were identified as non-route-specific as they have previously been identified in MDA synthesised from 3,4-methylenedioxycinnamic acid or piperonal. These non-route-specific impurities were also identified in MDA synthesised using sodium hypochlorite. No impurities were detected in MDA hydrochloride. This study identified route- and condition-specific organic impurities in MDA synthesised via the "twodogs" synthetic route using helional as starting material. The results in this study provide further understanding into the illicit synthesis of MDA and highlight the expanding nature of precursors used for illicit drug manufacture. It provides valuable information to decision makers to enact legislative measures and restrict precursors of concern., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Nearly a decade ago, fentanyl reappeared in the United States illicit drug market. In the years since, overdose deaths have continued to rise as well as the amount of fentanyl seized by law enforcement agencies. Research surrounding fentanyl production has been beneficial to regulatory actions and understanding illicit fentanyl production. In 2017, the Drug Enforcement Administration (DEA) began collecting seized fentanyl samples from throughout the United States to track purity, adulteration trends, and synthetic impurity profiles for intelligence purposes. The appearance of a specific organic impurity, phenethyl-4-anilino-N-phenethylpiperidine (phenethyl-4-ANPP) indicates a shift in fentanyl production from the traditional Siegfried and Janssen routes to the Gupta-patent route. Through a collaboration between the DEA and the US Army's Combat Capabilities Development Command Chemical Biological Center (DEVCOM CBC), the synthesis of fentanyl was investigated via six synthetic routes, and the impurity profiles were compared to those of seized samples. The synthetic impurity phenethyl-4-ANPP was reliably observed in the Gupta-patent route published in 2013, and its structure was confirmed through isolation and structure elucidation. Organic impurity profiling results for illicit fentanyl samples seized in late 2021 have indicated yet another change in processing with the appearance of the impurity ethyl-4-anilino-N-phenethylpiperidine (ethyl-4-ANPP). Through altering reagents traditionally used in the Gupta-patent route, the formation of this impurity was determined to occur through a modification of the route as originally described in the Gupta patent., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA.)

Cardiovascular disorders are among the leading causes of death worldwide, especially hypertension, a silent killer syndrome requiring multiple drug therapy for appropriate management. Hydrochlorothiazide is an extensively utilized thiazide diuretic that combines with several antihypertensive drugs for effective treatment of hypertension. In this study, sustainable, innovative and accurate high performance liquid chromatographic methods with diode array and tandem mass detectors (HPLC-DAD and LC-MS/MS) were developed, optimized and validated for the concurrent determination of Hydrochlorothiazide (HCT) along with five antihypertensive drugs, namely; Valsartan (VAL), Amlodipine besylate (AML), Atenolol (ATN), Amiloride hydrochloride (AMI), and Candesartan cilextil (CAN) in their diverse pharmaceutical dosage forms and in the presence of Chlorothiazide (CT) and Salamide (DSA) as HCT officially identified impurities. The HPLC-DAD separation was achieved utilizing Inertsil ODS-3 C 18 column (250 × 4.6 mm, 5 μm) attached with photodiode array detection at 225.0 nm. Gradient elution was performed utilizing a mixture of solvent A (20.0 mM potassium dihydrogen phosphate, pH 3.0 ± 0.2, adjusted with phosphoric acid) and solvent B (acetonitrile) at ambient temperature. Linearity ranges were 0.1-100.0 µg/mL for HCT, VAL, AML and CAN, 0.05 -100.0 µg/mL for both ATN and AMI and 0.05-8.0 µg/mL for both CT and DSA. Additionally, this work describes the use of liquid chromatography-electrospray-tandem mass spectrometry for the accurate detection and quantification of the impurities; CT and DSA in the negative mode utilizing triple quadrupole mass spectrometry. The linearity ranges for those impurities were 1.0-200.0 ng/mL and 5.0-200.0 ng/mL for CT and DSA, respectively. Developed methods' validation was achieved in accordance with International Conference on Harmonization (ICH) guidelines. Upon applying liquid chromatographic techniques for the drug analysis, a green and sustainable assessment have to be handled due to the consumption of energy and many solvents. Through the use of the HEXAGON, Analytical Greenness (AGREE) and White Analytical Chemistry (WAC) tools, greenness and sustainability have been statistically assessed. The optimized HPLC-DAD and LC-MS/MS methods were fast, accurate, precise, and sensitive, and consequently could be applied for conventional analysis and quality control of the proposed drugs in their miscellaneous dosage forms for the purpose of reducing laboratory wastes, time of the analysis time, effort, and cost., (© 2023. Springer Nature Switzerland AG.)

A previously developed high-performance liquid chromatography method combined with pulsed amperometric detection allowed to separate many impurities of paromomycin. However, due to the presence of ion pairing agents and sodium hydroxide in the mobile phase, direct coupling to mass spectrometry for the identification of the chemical structures of the impurities was not an option. Indeed, ion suppression was encountered by trifluoroacetic acid and pentafluoroproponic acid in the mobile phase. A cation self-regenerating suppressor, which was originally designed for increasing analyte conductivity of ammonia and amines analysis in ion chromatography, was coupled between the liquid chromatography and ion trap-time of flight-mass spectrometry and almost all trifluoroacetic acid and pentafluoroproponic acid in the mobile phase was removed. The limit of detection of paromomycin in this integrated system improved significantly to 20 ng/ml (0.4 ng). The chemical structures of 19 impurities were elucidated and seven impurities were reported for the first time., (© 2023 Wiley-VCH GmbH.)

Nowadays, ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the dominating generic method for the analysis of nucleic acid related compounds, such as antisense-oligonucleotides (ASO), small-interfering ribonucleic acid (siRNA) or other DNA or RNA type molecules and their conjugates. Despite of its effective performance, the usage of a high concentration of ion-pairing reagent in the eluent in IP-RPLC is unfavorable for the hyphenation with mass spectrometry (MS) which is required for a detailed structural characterization of the analytes and their structurally related impurities. In this work, we tested a polybutylene terephthalate (PBT)-bonded silica-based stationary phase for the separation of generically synthesized Patisiran as siRNA (antisense and sense single strands as well as their annealed double strand) giving some unexpected selectivity without any presence of ion-pairing reagents. Important chromatographic conditions affecting the separation have been investigated and evaluated. Furthermore, MS and tandem MS (MS/MS) characterization was possible without contamination of the MS system with ion-pair agent and related problems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Identification and characterization of biproducts/ impurities present in agrochemicals are critical in view of their efficacy and safety towards public health. We herein present our study on identification and characterization of an impurity, 5-chloro-2-cyano-N,N-dimethyl-4-p-tolylimidazole-1-sulfonamide (2) present in the fungicide, "cyazofamid". Intermittent HPLC analysis of the reaction of substituted imidazole (1) with N,N-dimethylsulfamoyl chloride suggested that 2 is formed during the reaction. Isolation by preparative HPLC and characterization by NMR, LC/HRMS, MS/MS and single crystal XRD analysis confirmed 2 as an isomer of cyazofamid, wherein the N,N-dimethyl sulfonamide group was positioned on the other nitrogen of imidazole in close proximity to chloride group. Computational studies further supported the formation of 2 and ruled out the other possible isomeric structures., (© 2023 Wiley-VCH GmbH.)

The objective of the proposed work was to develop a rapid and new reverse phase ultra-performance liquid chromatographic (RP-UPLC) method for the simultaneous quantification of related impurities of ipratropium bromide and salbutamol sulfate in the combined inhalation dosage form. Herein, the chromatographic separation was achieved on Acquity BEH C18 (100mm×2.1mm, 1.7μm) column by following gradient elution of solvent A as 2mM potassium dihydrogen phosphate with 0.025% of 1-pentane sulphonic acid sodium salt (pH 3.0 buffer) and solvent B as pH 3.0 buffer, acetonitrile and methanol in the ratio of (32:50:18, v/v/v) at a flow rate of 0.3mL/min. The samples were detected and quantified at 220nm. To prove the stability-indicating potential of the method, forced degradation studies were performed using acidic, basic, oxidative, thermal, and photolytic conditions. After sufficient exposure, the resultant solutions were injected and found that all degradants and impurities formed during stress studies were well separated from each other and from the main peak compounds. The performance of the method was validated according to the present ICH Q2 (R1) guidelines. The method has good linearity (r≥0.999) and consistent recoveries were obtained with a range of 91.3-108.8% for all compounds. The % RSD obtained for the precision experiments was less than 5% and also there is a good sensitivity (LOQ≤0.5μg/mL) for all compounds. The intended method proved its applicability and that it can be beneficial to pharmaceutical industries for quick quantification of related impurities and assay in quality control department for analysis of ipratropium bromide and salbutamol sulfate inhalation dosage form., (Copyright © 2022. Published by Elsevier Masson SAS.)

Apalutamide, an androgen receptor inhibitor, is used to treat prostate cancer. A stability-indicating high-performance liquid chromatography method was developed for the estimation of assay and organic impurities of apalutamide in drug substance and in tablet dosages using Design of Experiments. The chromatographic separation was achieved within 30 min using Atlantis dC 18 , 100 × 4.6 mm, 3.0 μm and the binary gradient program (10 mm KH 2 PO 4 , pH 3.5; acetonitrile). The detection wavelength, flow rate, column temperature and injection volume used were 270 nm, 1.0 ml/min, 45°C and 10 μl, respectively. The interaction of independent variables (pH, column temperature and flow rate) and their influences on HPLC parameters were studied using a central composite design, and then the peak separation and elution behaviors between apalutamide and its seven impurities were determined. The method validation was performed for linearity, detection limit, quantitation limit, accuracy, precision and robustness as per the International Conference on Harmonization. A high-quality recovery with good precision (91.7-106.0%) and correlations (r 2  > 0.997) within a linear range of 0.12-2.24 μg/ml (0.05-0.3%, w/w) were achieved consistently for assay and organic impurities of apalutamide. The stability-indicating characteristics of the proposed method were assessed through forced degradation and mass balance studies. An effort was made to figure out the chemical structures of newly formed degradation products (DP1-DP5) using LC-MS/MS., (© 2022 John Wiley & Sons Ltd.)

A systematic literature survey published in several journals of pharmaceutical chemistry and of chromatography used to analyze impurities for most of the drugs that have been reviewed. This article covers the period from 2016 to 2020, in which almost of chromatographic techniques have been used for drug impurity analysis. These chromatography techniques are important in the analysis and description of drug impurities. Moreover, some recent developments in forced impurity profiling have been discussed, such as buffer solutions, mobile phase, columns, elution modes, and detectors are highlighted in drugs used for the study. This primarily focuses on thorough updating of different analytical methods which include hyphenated techniques for detecting and quantifying impurity and degradation levels in various pharmaceutical matrices.

Forced degradation studies have been used to simplify analytical methodology development and achieve a deeper knowledge about the inherent stability of active pharmaceutical ingredients (API) and drug products. This provides insight into degradation species and pathways. Identification of impurities in pharmaceutical products is closely related to the selection of the most appropriate analytical methods like HPLC-UV, LC-MS/MS, LC-NMR, GC-MS, and capillary electrophoresis. Herein, recent trends in analytical perspectives during 2018-April 14, 2021, are discussed based on forced and impurity degradation profiling of pharmaceuticals. Literature review showed that several methods have been used for experimental design and analysis conditions such as matrix type, column type, mobile phase, elution modes, detection wavelengths, and therapeutic category. Thus, since these factors influence the separation and identification of the impurities and degradation products, we attempted to perform a statistical analysis for the developed methods according to the abovementioned factors.

Even though the impurity analysis of the Ph. Eur. is considered well-studied, its methodology should be reviewed periodically to ensure that it is working properly, and all impurities are captured by the section "Related substances" of the monograph. Within this study, the biotechnological produced antidiabetic drug acarbose was chosen to demonstrate some of the advantages as well as the shortcomings arising from the current related substances test of acarbose. Due to its weak chromophore, acarbose is studied by UV detection at 210 nm after being separated on aminopropyl-silyl stationary phases. Thus, the use of alternative detection techniques, such as charge aerosol detection (CAD) and a volatile mobile phase can be beneficial here. Since a simple method transfer to a mobile phase usable with the CAD was not possible, more stable stationary phases were tested. For the rapid determination of the sum of impurities, a method was developed using a pentafluorophenyl column and a mobile phase of 0.1% TFA in water. Maltose and maltotriose were further identified as additional impurities of the API. Furthermore, a method was developed and validated by means of an Amide-HILIC phase, that adequately separated acarbose and all of its impurities. However, the sensitivity of this method needs to be further improved. Additionally, a method was also developed and validated, taking into account the temperature stability of graphite columns. Thus, the separation was carried out at temperatures of about 90 °C to solve the problem of anomerization of acarbose and some of its impurities. In comparison with the other developed methods, the elution order was changed and has to be confirmed, but all components were separated and detected at a sufficient LOQ of 0.10%., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Ion-pair liquid chromatography with pulsed electrochemical detection (LC-PED) was established for the analysis of impurities in arbekacin (ABK) sulfate. APursuit pentafluorophenylpropyl (PFP) column was used as stationary phase. This novel method showed greater separation and sensitivity ability. In a representative ABK sample, 24 impurity peaks were detected in LC-PED, where of only 9 were monitored by a post-column derivatization method prescribed by the Japanese Pharmacopoeia (JP). For identification of the chemical structures of the impurities detected by LC-PED, LC-Mass Spectrometry (MS) was used. Two challenges had to be overcome in this work. The first was the transfer of the MS incompatible mobile phase to an MS compatibleone while maintaining the elution order of the peaks in the chromatograms. Previously reported approaches such as two-dimensional (2D)LC were hardly applicable in this case due to the lack of ultraviolet (UV) absorbing chromophores in ABK and its impurities. The sodium hydroxide solution was replaced by aqueous ammonia to adjust the pH of the mobile phase used in LC-PED. The other challenge encountered was the ion suppression effect caused by trifluoroacetic acid (TFA) and pentafluoroproponic acid (PFPA) in the mobile phase. Some strategies such as "TFA-fixed" and its modifications were tried, but they were inconvenient and severe contamination of the MS was observed. A cationself-regenerating suppressor (CSRS), which was originally designed for increasing analyte conductivityof ammonia and amines analysis in ion chromatography (IC), was coupled between the LC and Ion Trap-Time of Flight (IT-TOF)-MS and almost all TFA and PFPA in the mobile phase were removed. The limit of detection (LOD) of ABK in this integrated system improved significantly to 20 ng/mL. The chemical structures of the 28 impurities were elucidated and 15 impurities were reported for the first time., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Amitriptyline hydrochloride is an antidepressant drug with sedative effects used to treat the symptoms of anxiety, agitation with depression and schizophrenia with depression. A reversed-phase high-performance liquid chromatography method was developed to separate and quantitatively determine the assay and four organic impurities of amitriptyline in tablet dosage form and bulk drugs using a C 18 column in an isocratic elution mode with mobile phase consisting of a mixture of pH 7.5 phosphate buffer and methanol. The pH conditions used in the chromatographic separation are discussed. The stability-indicating characteristics of the proposed method were proved using stress testing [5 m HCl at 80°C/1 h, 5 m NaOH at 80°C/1 h, H 2 O (v/w) at 80°C/1 h, 6% H 2 O 2 (v/v) at 25°C/1 h, dry heat at 105°C/24 h and UV-vis light/4 days] and validated for specificity, detection limit, quantitation limit, linearity, precision, accuracy and robustness. For amitriptyline and its four known organic impurities, the quantitation limits, linearity and recoveries were in the ranges 0.25-3.0 μg/ml (r 2  > 0.999) and 87.9-107.6%, respectively. The mass (m/z) spectral data of amitriptyline hydrochloride and its impurity are discussed. The proposed LC method is also suitable for impurity profiling and assay determination of amitriptyline in bulk drugs and pharmaceutical formulations., (© 2022 John Wiley & Sons Ltd.)

Dinotefuran (DNT) is a neonicotinoid insecticide widely used in pest control. Identification of structurally related impurities is indispensable during material purification and pesticide registration and certified reference material development, and therefore needs to be carefully characterized. In this study, a combined strategy with liquid chromatography high-resolution mass spectrometry and SIRIUS has been developed to elucidate impurities from DNT material. MS and MS/MS spectra were used to score the impurity candidates by isotope score and fragment tree in the computer assisted tool, SIRIUS. DNT, the main component, worked as an anchor for formula identification and impurity structure elucidation. With this strategy, two by-product impurities and one stereoisomer were identified. Their fragmentation pathways were concluded, and the mechanism for impurity formation was also proposed. This result showed a successful application for combined human intelligence and machine learning, in the identification of pesticide impurities.

The group of P2P precursors including α-phenylacetoacetonitrile (APAAN), α-phenylacetoamide (APAA) and methyl α-acetylphenylacetate (MAPA) has become increasingly popular in Europe and other parts of the world in the last decade. Previous investigations have reported the use of APAAN in the synthesis of amphetamine and methamphetamine and identified a range of characteristic impurities. This research has expanded upon the current literature by investigating the use of MAPA in the synthesis of methamphetamine. In this study methamphetamine was synthesized via three common clandestine methods: the Leuckart synthesis and two reductive amination methods. We report the identification of seven impurities, four of which are methyl ester equivalents of impurities previously reported for the detection of APAAN. These are methyl 2-phenylbut-2-enoate, methyl 2-phenyl-3-hydroxybutanoate, methyl 3-(methylamino)-2-phenylbut-2-enoate and methyl 3-(methylamino)-2-phenylbutanoate. The other impurities identified are ethyl ester compounds formed via transesterification of the methyl ester due to the reaction solvent. This susceptibility for transesterification suggests that identification of the pre-precursor used may not always be straightforward and may be dependent on the reaction conditions employed. Of the impurities reported, methyl 3-(methylamino)-2-phenylbutanoate was deemed to be a potentially reliable impurity for detection of the use of MAPA; however, it is expected that lower levels of characteristic impurities may be detected in methamphetamine synthesized from MAPA than that from APAAN., (© 2022 John Wiley & Sons, Ltd.)

In 2018, the discovery of carcinogenic nitrosamine process related impurities (PRIs) in a group of widely used drugs led to the recall and complete withdrawal of several medications that were consumed for a long time, unaware of the presence of these genotoxic PRIs. Since then, PRIs that arise during the manufacturing process of the active pharmaceutical ingredients (APIs), together with their degradation impurities, have gained the attention of analytical chemistry researchers. In 2020, favipiravir (FVR) was found to have an effective antiviral activity against the SARS-COVID-19 virus. Therefore, it was included in the COVID-19 treatment protocols and was consequently globally manufactured at large-scales during the pandemic. There is information indigence about FVR impurity profiling, and until now, no method has been reported for the simultaneous determination of FVR together with its PRIs. In this study, five advanced multi-level design models were developed and validated for the simultaneous determination of FVR and two PRIs, namely; (6-chloro-3-hydroxypyrazine-2-carboxamide) and (3,6-dichloro-pyrazine-2-carbonitrile). The five developed models were classical least square (CLS), principal component regression (PCR), partial least squares (PLS), genetic algorithm-partial least squares (GA-PLS), and artificial neural networks (ANN). Five concentration levels of each compound, chosen according to the linearity range of the target analytes, were used to construct a five-level, three-factor chemometric design, giving rise to twenty-five mixtures. The models resolved the strong spectral overlap in the UV-spectra of the FVR and its PRIs. The PCR and PLS models exhibited the best performances, while PLS proved the highest sensitivity relative to the other models.

Sarin is a highly toxic nerve agent classified by the Chemical Weapon Convention as a Schedule 1 chemical with no use other than to kill or injure. Moreover, in recent times, chemical warfare agents have been deployed against both military and civilian populations. Chemical warfare agents always contain minor impurities that can provide important chemical attribution signatures (CAS) that can aid in forensic investigations. In order to understand the trace molecular composition of sarin, various analytical approaches including GC-MS, LC-MS and NMR were used to determine the chemical markers of a set of sarin samples. Precursor materials were studied and the full characterisation of a synthetic process was undertaken in order to provide new insights into potential chemical attribution signatures for this agent. Several compounds that were identified in the precursor were also found in the sarin samples linking it to its method of preparation. The identification of these CAS contributes critical information about a synthetic route to sarin, and has potential for translation to related nerve agents., (© 2022. Crown.)

Montelukast sodium (MLS) is a leukotriene receptor antagonist drug used in the treatment of asthma, bronchospasm, allergic rhinitis and urticaria. A reversed-phase high performance liquid chromatography method was developed to separate, identify and quantitative determination of MLS and its eight known organic impurities in tablet dosage form using a C 18 column and mobile phases consisting of a gradient mixture of pH 2.5 phosphate buffer and acetonitrile. The stability-indicating character of the developed method was proven using stress testing (1 m HCl at 80°C/30 min, 1 m NaOH at 80°C/30 min, H 2 O at 80°C/30 min, 3% H 2 O 2 at 25°C/1 min, dry heat at 105°C/10 h and UV-vis light/4 days) and was validated for specificity, quantitation limit, linearity, precision, accuracy and robustness. For MLS and its eight known impurities, the quantitation limits, linearity and recoveries were 0.015-0.03 μg/ml, correlation coefficient > 0.997 (R 2  > 0.995) and 85.5-107.0%, respectively. The developed chromatographic method is suitable for impurity profiling and also for assay determination of MLS in bulk drugs and pharmaceutical formulations. The mass values (m/z) of newly formed degradation products (DP1 and DP2) of montelukast sodium were identified using liquid chromatography-mass spectrometry., (© 2022 John Wiley & Sons, Ltd.)

An HPLC method has been described in the European Pharmacopoeia and United States Pharmacopeia for the determination of nine organic impurities (imp A-I) in fingolimod hydrochloride, a synthetic sphingosine-1-phosphate receptor modulator. The manufacturing process of fingolimod hydrochloride consists of multistep chemical synthesis wherein controls of precursors, intermediates and process steps should be performed to assure the final quality of the drug substance. We synthesized and isolated eight process-related impurities (FINI imp A-H) of fingolimod, which were different from the pharmacopoeial impurities. One unknown process-related impurity was found as a key intermediate (FINI) and was identified by LC-MS. Characterization of all of the impurities were done using spectroscopic techniques ( 1 H and 13 C NMR, FTIR, MS), and the mechanistic pathways to the formation of these impurities were also discussed. Two of these impurities were evaluated as potential genotoxic impurities owing to their alerting structures and alkylating properties (alkyl sulfonates and alkyl halides, class 3, ICH M7). We also developed and validated an RP-UPLC method in line with ICH Q2 guidelines for control these impurities (FINI imp A-H) and to assure the pharmacopoeial quality drug substance., (© 2022 John Wiley & Sons, Ltd.)

N-Rich is a twin-column chromatography process that enriches target compounds relative to other components in a mixture, thereby facilitating their isolation and characterization. This study demonstrates the performance of N-Rich for isolation of Angiotensin II peptide impurities compared with standard analytical and preparative chromatography approaches. Peptides have diverse chemical properties and are produced using a wide range of methods, resulting in products with complex impurity profiles. The characterization of impurities for clinical development is essential but obtaining high purity samples in sufficient quantities is often a difficult task when using standard chromatographic techniques. In contrast, by using cyclic continuous chromatography with UV-based process control, N-Rich enables automatic on-column accumulation of target impurities while other compounds in the mixture are depleted. This has multiple advantages compared to standard techniques. Firstly, at the end of the cyclic accumulation phase the highly enriched target is eluted in one step with high purity and concentration. This means fewer fractions for analysis are generated and up-concentration steps are reduced. Secondly, the purification of target impurities using semi-preparative scale chromatography becomes viable, even if initial resolution is poor compared to analytical HPLC. This allows for very significant increases in productivity for purification of difficult to isolate impurities. This study demonstrates two N-Rich strategies: Example 1: Purification of µg quantities of multiple Angiotensin II impurities with a >9-fold increase in productivity compared to analytical HPLC. Example 2: Specific isolation of 1 mg of a critical impurity at 88% purity. 79-fold increase in productivity and a 69-fold reduction in solvent consumption compared to analytical HPLC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Impurity profiling has a rising importance nowadays due to the increased health problems associated with impurities and degradation products found in several drug substances and formulations. Three advanced, accurate and precise chemometric methods were developed as impurity profiling methods for a mixture of bisoprolol fumarate (BIS) and perindopril arginine (PER) with their degradation products which represent drug impurity or a precursor to such impurity. The methods applied were Partial Least Squares (PLS-1), Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) and Artificial Neural Networks (ANN). Genetic Algorithm (GA) was used as a variable selection tool to select the most significant wavelengths for the three chemometric models. For proper analysis, a 5-factor 5-level experimental design was used to establish a calibration set of 25 mixtures containing different ratios of the drugs and their degradation products (impurities). The validity of the proposed methods was assessed using an independent validation set. The designed models were able to predict the concentrations of the drugs and the degradation products/impurities in the validation set and pharmaceutical formulation. The proposed methods presented a powerful alternative to traditional and expensive chromatographic methods as impurity profiling tools., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Capillary electrophoresis (CE) is widely used for the impurity profiling of drugs that contain stereochemical centers in their structures, analysis of biomolecules, and characterization of biopharmaceuticals. Currently, CE is the method of choice for the analysis of foodstuffs and the determination of adulterants. This article discusses the general theory and instrumentation of CE as well as the classification of various CE techniques. It also presents an overview of research on the applications of different CE techniques in the impurity profiling of drugs in the past decade. The review briefly presents a comparison between CE and liquid chromatography methods and highlights the strengths of CE using drug compounds as examples. This review will help scientists, fellow researchers, and students to understand the applications of CE techniques in the impurity profiling of drugs., Competing Interests: The authors declare that there are no conflicts of interests., (© 2021 Xi'an Jiaotong University. Production and hosting by Elsevier B.V.)

The impurity profile of budesonide solution-formulated metered dose inhalation using thermal forced degradation approach was studied intensively in this article. The structural identification of 10 budesonide related impurities was conducted by LC-QTOFMS, and the impurity level in the formulations of different excipients and packing materials were compared using HPLC-UV. Based on our results, the impurities were classified into three groups: (Ⅰ) process impurities, including budesonide impurity A, C and F; (Ⅱ) degradation products, including budesonide impurity E, G, D, 17-carboxylate, and 17-ketone; (Ⅲ) not only process impurities but also degradation products, including budesonide impurity I and L. Budesonide impurity D, 17-carboxylate, 17-ketone and impurity L were found to be the major degradation products of budesonide, and the reaction pathways for the generation of these impurities were speculated. The generation of budesonide impurity D, 17-carboxylate and L was found to be an aerobic oxidation process induced by Al 2 O 3 on the inner surface of aluminum canisters. Furthermore, an in-depth discussion on the proposed impact of the excipients on budesonide degradation, especially on the Al 2 O 3 -induced oxidation process, was provided in this article., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cocaine is one of the most widely used illicit drugs worldwide. Cocaine powders seized by the Police may contain numerous other substances besides the drug itself. These can be impurities originating from the coca plant or the production process, or be purposely added to the drug formulation as adulterants and cutting agents. In forensic laboratories, identification of cocaine is routinely done through GC-MS analysis, but other components are often ignored even if the method allows for their detection. Yet, they can provide valuable insight into the history of a seizure and its potential connection to other samples. To explore this idea, an extensive review of common impurities and adulterants encountered in cocaine is presented. Based on their incidence, concentration in the end product and compatibility with GC-MS methods, their overall usefulness as candidates for the statistical investigation of existing forensic data is evaluated. The impurities cis- and trans-cinnamoylcocaine, tropacocaine, norcocaine and N-benzoylnormethylecgonine as well as the adulterants lidocaine, procaine, tetracaine, benzocaine, caffeine, acetylsalicylic acid, phenacetin, ibuprofen, levamisole, hydroxyzine and diltiazem are promising candidates to provide additional forensic intelligence. Future research on optimized routine GC-MS methods, signal reproducibility, comparison, statistics and databases is suggested to facilitate this concept. Ultimately, such an approach may significantly advance the amount of information that is extracted from routine casework data, elucidate developments in the cocaine markets in the past and facilitate Police work in the future. Preliminary assessment of existing data from the forensic laboratory of the Amsterdam Police has been included to show that the detection of the identified target impurities is feasible, and that small adjustments to the analysis method could significantly increase the detectability of these analytes in prospective drug screenings. Forensic intelligence based on retrospective data mining of cocaine containing casework samples may thus be realized with minimal additional laboratory efforts by using already available instrumentation, samples and data., (Copyright © 2021 The Chartered Society of Forensic Sciences. Published by Elsevier B.V. All rights reserved.)

The rise in popularity of 'designer' precursor compounds for the synthesis of amphetamine-type stimulants poses a significant challenge to law enforcement agencies. One such precursor is α-phenylacetoacetonitrile (APAAN). APAAN emerged in Europe in 2010 and quickly became one of the most popular precursors for amphetamine synthesis in that region. Previous literature has identified four APAAN-specific impurities formed in the synthesis of amphetamine; however, there is currently no research on the use of APAAN in the synthesis of methamphetamine, which is more likely to be employed in a non-European market. In this study methamphetamine was synthesised via three common clandestine methods: the Leuckart method and two reductive amination methods. We report the identification of five new impurities and two previously identified impurities characteristic for the use of APAAN in the synthesis of methamphetamine. The newly identified impurities were characterised by MS and NMR and determined to be (E)-3-(methylamino)-2-phenylbut-2-enenitrile, 3-(methylamino)-2-phenylbutanenitrile, 3-methyl-2,4-diphenylpentanedinitrile, 2-phenylbutyronitrile and 3-hydroxy-2-phenylbutanenitrile., (© 2021 John Wiley & Sons, Ltd.)

Midostaurin (MDS) is used for the treatment of acute myeloid leukemia, myelodysplastic syndrome, and advanced systemic mastocytosis. MDS softgel capsule samples were subjected to stress testing per International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines for impurity profiling study. MDS underwent extensive degradation under stress testing (acid, alkaline, oxidative, photolytic, thermolytic, and hydrolysis conditions) and formed four degradation products (DPs). MDS and its DPs were separated well from one another with good resolution using reserved-phase HPLC using an Inertsil ODS-3V column (250 × 4.6 mm, 5 μm) and a mobile phase of ammonium formate (40 mM) and acetonitrile. The stability-indicating characteristic of the newly developed method was proven for the estimation of MDS assay, and its organic impurities were free from interference. The validated method exhibited excellent linearity, accuracy, precision, specificity, detection limit, and quantitation limit within 25 min run time. Stress testing, robustness, and solution stability were performed to ensure the continuous performance of the developed method. The peak fractions of DPs formed under stress testing were isolated and characterized using LC-MS, 1 H and 13 C NMR, IR, and UV-Vis. The structure of the major DPs was predicted as DP1 based on the spectral data. The proposed method is effectively used for MDS in bulk drug and finished formulations in the pharmaceutical industry., (© 2021 John Wiley & Sons, Ltd.)

The main objective of the study is to develop a suitable and rapid UPLC/PDA method by coupling online to Quadrupole Dalton analyzer (QDa), a mass detector for the identification and impurity profiling of Brimonidine Tartrate (BRIM)/Timolol maleate (TIMO) in the ophthalmic formulation. Chromatographic separation was achieved on ethylene bridged hybrid octadecylsilane column having 1.7μm particle size in gradient mode using high pure heptafluorobutyric acid as a buffer (A) and water, methanol, and acetonitrile (B) as mobile phase with a flow rate of 0.3mlmin -1 . Based on the spectral maxima, BRIM and its impurities were monitored at 248nm, and TIMO and its impurities were monitored at 295nm. During evaluation of stress conditions and stability data unknown degradants are observed and identified as m/z 218.01 (DP1) and m/z 390.03 (DP2) using QDa-ESI+ scanning mode technique. The performance of the method was systematically validated according to ICH Q2 (R1) guidelines and the method shown very good sensitivity (≥0.5μg.mL -1 ) and linearity (r 2 ≥0.999) with consistent recoveries and less than 5% RSD for all compounds. Hence, the proposed UPLC/PDA/QDa method is a simple, sensitive and comprehensive technique where identification and quantification can be done. It gives for complete impurity profile evaluation of BRIM/TIMO in the ophthalmic formulation during quality control in the pharmaceutical industry., (Copyright © 2021 Académie Nationale de Pharmacie. Published by Elsevier Masson SAS. All rights reserved.)

A new RP-HPLC method with a quick, sensitive and stable indication for the quantitative measurement of selexipag and its associated substances was developed and validated in the present study. In this new method, using the impurity-spiked solution, the chromatographic approach was optimized. Similarly, using the X-bridge phenyl column with isocratic elution of mobile phase containing acetonitrile and formic acid, selexipag and its impurities were separated. Recovery experiments obtained were satisfactory, and also the calibration graphs plotted for selexipag and its five impurities were found to be linear. The system validation parameters such as specificity, linearity, precision, accuracy and robustness were determined successfully. The obtained results indicated that the developed method was found to be useful for analyzing selexipag from its impurities. Further, using stress tests against acid, alkali, peroxide, reduction, thermal, hydrolysis and UV conditions, the present established method of HPLC was assessed and validated as per ICH Q2(R1) guidelines., (© 2021 John Wiley & Sons, Ltd.)

Upon emergence of nitrosamines in various drugs, e.g in valsartan, metformin and ranitidine, 4-methyl-1-nitrosopiperazine (MeNP) was found in rifampicin in August 2020. Rifampicin is used, amongst others, for post-exposure prophylaxis of leprosy. The occurrence of MeNP can be explained by the synthesis, because 1-amino-4-methylpiperazine is concomitantly used with the organic oxidizing reagent isoamyl nitrite. According to a method reported by the FDA, the quantification of MeNP in rifampicin capsules was performed by LC-MS/HRMS. A significant contamination with MeNP was found in all samples, ranging from 0.7 to 5.1 ppm and exceeding the acceptable intake limit proposed by the FDA up to 32-fold. However, the severity of a possible leprosy infection outweighs the risks, which are concomitant with the intake of a single dose of rifampicin for post-exposure prophylaxis. Nevertheless, the extent of contamination is alarming, and countermeasures are needed to minimize public health risks. The presence of nitrosamines in rifampicin illustrates the need for better strategies in impurity profiling and compendial testing once again., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Two selective, sensitive and environmentally safe LC methods were developed and validated for determination of paracetamol, caffeine, ergotamine tartrate and metoclopramide in coformulated antimigraine tablets along with p-aminophenol, p-nitrophenol and theophylline as officially specified impurities. The first is based on high-performance thin-layer chromatography (HPTLC) coupled with densitometric quantitation. Separation was achieved on HPTLC silica gel 60 F 254 plates as stationary phase using ethyl acetate:aqueous ammonium hydroxide solution:glacial acetic acid (10.0:0.4:0.1, by volume) as a developing system followed by scanning of the separated bands at 210.0 nm. The subsequent method depends on HPLC with diode array detection. The LC separation was accomplished on a Scharlau C 18 (250 × 4.6 mm, 5 μm) column using a mixture of 20.0 mm sodium dihydrogen phosphate, pH 3.0, adjusted with o-phosphoric acid and methanol, at a flow rate of 1.3 mL/min in a gradient elution program. The separated peaks were detected at 210.0 nm. The proposed methods have been validated and proven to meet the requirements outlined in the International Council for Harmonisation (ICH) guidelines. The greenness profile evaluation was carried out using three tools, namely, the National Environmental Method Index, the Analytical EcoScale and the Green Analytical Procedure Index tool, and a comparative study was then conducted. Successful application of the developed methods for determination of the cited quaternary mixture in Metograine tablets confirms their suitability regarding the analytical performance and ecological impact in quality control assay and impurity profiling purposes., (© 2021 John Wiley & Sons, Ltd.)

The quality control of active pharmaceutical ingredients (APIs) is a very important aspect for drug products entering the market. However, also for the well-established drugs, there ought to be a state-of-the-art impurity control. Some of the pharmacopoeial tests for related substances still make use of thin layer chromatography, even though selectivity and sensitivity are suboptimal. Here, we report on the development of a new gradient high performance liquid chromatography (HPLC) method for dapsone in order to replace the currently described pharmacopoeial TLC method. The separation of all relevant components was achieved on a C18 stationary phase (Waters XTerra® RP 18 5 μm 4.6 × 250 mm) using a water-acetonitrile gradient. A limit of detection (LOD) of 0.02% was registered for all specified impurities. Additionally, within this study an "impurity of an impurity" was identified by means of LC-MS/MS., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A short RNA with the sequence of the antisense strand of Patisiran has been selected as test material for the investigation of its common impurities using three different two-dimensional liquid chromatography (2D-LC) platforms. On the one hand, a quinine (QN) carbamate-based weak anion-exchange (AX) stationary phase (QN-AX) and a classical C18 reversed phase (RP) stationary phase in ion-pair (IP) mode with tripropylammonium acetate, respectively, have been used in the first dimension ( 1 D) to provide the selectivity for impurities formed during the synthesis of the RNA. In the next step, certain peaks of interest from 1 D have been transferred by multiple-heart-cutting (MHC) into a 2 D in which an ESI-MS-compatible non-ionpairing RP method has been used for desalting via a diverter valve to remove non-volatile phosphate buffer components and ion-pair agents, respectively. Thus, a sensitive electrospray-ionization quadrupole time of flight mass spectrometry (ESI-TOF-MS) analysis of resolved impurity peaks of the siRNA has become possible under MS-friendly conditions. With both 2D-LC setups, peak purity of the ON has been evaluated by selective comprehensive (high resolution) sampling of the main peak. In a third MHC 2D-LC approach, the QN-AX LC mode was online coupled with the IP-RPLC in the 2 D using UV detection. It allows the separation of additional impurities which coeluted in the first dimension. The potential of these methods for comprehensive impurity profiling of ON therapeutics is illustrated and discussed., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interests., (Copyright © 2021 Elsevier B.V. All rights reserved.)

From an analytical chemistry standpoint, determining the chemical attribution signatures (CAS) of synthetic reaction mixtures is an impurity profiling exercise. Identifying and understanding the impurity profile and CAS of these chemical agents would allow them to be exploited for chemical forensic information, such as how a particular chemical agent was synthesised. Being able to determine the synthetic route used to make a chemical agent allows for the possibility of batches of the agent, and individual incidents using that agent, to be forensically linked. This information is of particular benefit to agencies investigating the nefarious and illicit use of chemical agents. One such chemical agent of interest to law enforcement and national security agencies is fentanyl. In this study two acylation methods for the final step of fentanyl production, herein termed the Janssen and Siegfried methods, were investigated by liquid chromatography- high resolution mass spectrometry (LC-HRMS) and multivariate statistical analysis (MVA). From these data, fifty-five chemical impurities were identified. Of these, ten were specific CAS for the Janssen method, and five for the Siegfried method. Additionally, analytical data from four different literature methods for production of the fentanyl precursor 4-anilino-N-phenethylpiperidine (ANPP), were compared to the results obtained from the method of production (Valdez) used in this study. Comparison of the LC-HRMS data for these five methods allowed for four Valdez specific impurities to be identified. These may be useful CAS for the Valdez method of ANPP production., Competing Interests: Declaration of Competing Interest The authors declare that they have no know competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript., (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)

BMS-986142 has been developed as an innovative Bruton's tyrosine kinase inhibitor for treatment of several autoimmune diseases. The drug substance of BMS-986142 may contain three potential atropisomeric impurities due to its unique structural characteristics. Developing a single liquid chromatography (LC) method to separate all four highly structurally related atropisomers and other process impurities from each other turned out to be a daunting task. Two-dimensional LC (2DLC) was found to be an extremely powerful enabling technology for extracting purity information out of the complex sample impurity profile and facilitated process development before a final single dimension method was discovered. The off-the-shelf 2DLC instrument could be configured to allow injection of the targeted first dimension peak through either no-loss multiple heart-cutting fractions or as a large, single volume fraction with on-line dilution. Excellent precision (relative standard deviation of 0.3 %) and recovery (101.2 ± 0.2 %) was achieved for an atropisomer impurity at a 10 % monitoring level in the first configuration with sensitivity down to 0.2 % w/w. With the second instrument configuration, which eliminated the need for fraction recombination, similar figures of merit were maintained for the second dimension at the cost of losing the ability to collect and park multiple fractions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Profiling impurities for the active pharmaceutical ingredients (APIs) is an indispensable step in drug development process. Nowadays, high resolution mass spectrometry is the first choice for determining the chemical formula of organic impurities. However, merely base on the accurate mass to screen the formula is obviously not a flawless method. In this paper, a reliable strategy based on Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was presented to profile the related impurities. Firstly, Cefteram pivoxil was subjected to forced degration under hydrolytic (acidic and basic), oxidative, photolytic and thermal conditions according to ICH guidelines. Then, a highly specific and efficient HPLC-FT-ICR MS compatible method was developed and it was used to separate and characterize the process related substances and the major degradation products in Cefteram pivoxil. Next, isotopic fine structures (IFSs) of all impurities were acquired to decisively determine their elemental composition. Finally, the possible chemical structures of impurities were predicted by combining the information of accurate mass, retention time, IFSs and characteristic fragmentation ions. As a result, a total of 20 related substances including 6 process related substances and 14 degradation products were identified and characterized. To the best of our knowledge, 13 of these related substances were not reported in the previous literature. It indicates that the developed strategy is accurate and standard independent to determine the chemical formulae of organic impurities in APIs. In conclusion, the impurity profiles obtained in this study are critical to the quality control and stability study of Cefteram pivoxil. Moreover, the developed method can be used as a versatile workflow to profile the impurities in APIs in the future, especially for the unknown impurities., Competing Interests: Declaration of Competing Interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A methodology for the qualitative analysis of a mixture of compounds obtained during the synthesis of difluprednate is described herein for the first time. For this scope a multi-technique analytical approach was developed, combining Liquid Chromatography/Mass Spectrometry (LC/MS), Nuclear Magnetic Resonance (NMR) and computational chemistry. Separation of isomers is frequently required for the identification of impurities in active pharmaceutical ingredients (APIs) to assess the impact they may exhibit on public health. During the final step of the difluprednate synthesis apart from the desired product, various by-products may be obtained. Structural analysis of the products using LC/MS and NMR indicated that the steroid difluprednate was obtained along with its acetyl/butyryl regional isomers, whereas the results were further supported by semi-empirical calculations of the MS-derived data. Following the proposed approach, we managed to elucidate the structures of the challenging 11-acetate, 17-butyrate from the 17-acetate, 21-butyrate, 6α,9α-difluoro prednisolone isomers. The approach utilized may be of general applicability for the analysis of impurities in active pharmaceutical ingredients obtained during chemical synthesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

There is a huge, still increasing market for synthetic and therapeutic peptides. Their quality control is commonly based on a generic reversed-phase liquid chromatography (RPLC) method with C18 stationary phase and acetonitrile gradient with 0.1% trifluoroacetic acid in the mobile phase. It performs exceptionally well for a wide variety of impurities, yet structurally closely related impurities with similar sequences, not resolved in preparative RPLC, may easily coelute in the corresponding QC run as well. To address this problem an advanced generic 2D-LC impurity profiling method was developed in this work. It employs a selective comprehensive (high resolution sampling) RP×RP 2D-LC separation using a 100×2.1 mm ID column with the common acidic generic gradient in the first dimension, while RPLC under basic pH on a short 30×3 mm ID column is used in the second dimension. Recording data with a UV detector at 215 nm after 1 D separation provides the common generic 1D chromatogram. However, after the 2 D separation a flow splitter enabled recording of the signals of complementary detectors comprising a diode array detector (DAD) in-line with a charged aerosol detector (CAD) and a quadrupole-time-of-flight (QTOF) mass spectrometer (MS) with an electrospray ionization (ESI) source. Generic conditions of this 2D-LC method have been established through optimization of 2 D stationary and mobile phase considering different pH values and buffer concentrations. The orthogonal separation principle has been documented by a number of therapeutic peptides including Exenatide, Octreotide, Cyclosporine A and Oxytocin as well as some other proprietary synthetic peptides. The information density can be further enhanced by using the QTOF-MS detector by data independent acquisition with SWATH. Through this sequential window acquisition of all theoretical fragment ion mass spectra it became possible to collect MS/MS data comprehensively in the high-resolution sampling window, thus enabling the extraction of 2D-EICs from fragment ions and the generation of 2D-contour plots of all product ions. Using Oxytocin as an example for an important therapeutic peptide, the ability of this advanced generic sRP-UV×RP-DAD-CAD-ESI-QTOF-MS/MS method with SWATH for peptide quality control is discussed., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interests., (Copyright © 2020. Published by Elsevier B.V.)

Currently, analytical scientists are paying special attention to reducing reliance on hazardous chemicals in various analytical methods. By embracing this concept, we developed an eco-friendly high-performancethin-layer chromatography (HPTLC) method as an alternative for the conventional HPLC method for the determination of an essential human micronutrient, niacin (NIA), which is used improve the lipid profile of patients. Furthermore, the proposed HPTLC method is capable of determining the structurally related impurities of NIA such as pyridine-2,5-dicarboxylic acid, isonicotinic acid, pyridine, and 5-ethyl-2-methylpyridine, which exhibit nephrotoxic and hepatotoxic effects. The separation of this challenging mixture was achieved on HPTLC sheets using a mixture of ethyl acetate/ethanol/ammonia solution (6:4:0.05, v/v/v), and then the dried plates were scanned at 254 nm. The analytical eco-scale assessment protocol was used to assess the greenness profile of the presented method and compare it with the reported HPLC method. The suggested method was found to be greener with regard to the consumption of solvents and the yielding of waste. The results suggest that the described method can be safely implemented for the routine analysis of NIA pharmaceutical dosage without the interference of potential impurities in quality control laboratories., (© 2020 John Wiley & Sons, Ltd.)

For the compendial related substances test of l-aspartic acid (Asp) and glycine (Gly), two separate reversed-phase ion-pair high-performance liquid chromatography methods coupled with charged aerosol and ultraviolet detection were developed. Separation of all putative impurities, in particular of the related carboxylic and amino acids, was achieved using volatile perfluorocarboxylic acids as ion-pairing reagents on a polar embedded C18 stationary phase. It was shown that an adjustment of the evaporation temperature of the charged aerosol detector (CAD) was an efficient strategy for meeting the required quantitation limits, when dealing with non-volatile analytes. It was also demonstrated that the usage of a two detector setup can be beneficial for extending the detection range and providing accurate quantitation of low level impurities (LOQs from 5 to 50 ng on column). Both methods were validated with accordance to ICH guideline Q2(R1) assessing specificity, linearity, accuracy, precision, and robustness. Several batches of Asp and Gly were tested for related substances using the developed methods. The purity of each sample was higher than 99.7 %. Coupled charged aerosol and UV detection proved to be a more simple, robust and selective alternative to established derivatization procedures such as the Amino-Acid-Analyser (AAA) for the impurity profiling of amino acids, and should thus be considered for implementation into pharmacopoeial monographs in the future., Competing Interests: Declaration of Competing Interest None of the authors of this paper does have a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The organophosphorous nerve agent VX is classified by the Chemical Warfare Convention (CWC) as a Schedule 1 chemical; namely a substance that is highly toxic with no use that is of benefit to society. Even with this classification, the nefarious use of the Schedule 1 chemical VX has been observed, as demonstrated in 2017 in Malaysia. Therefore, undertaking chemical analysis on samples of VX to identify chemical attribution signatures (CAS) for chemical forensics is required. To further understand the chemical profile of VX, and to aid in the identification of potential CAS, three in house synthesised stocks of VX were investigated. The three VX stocks analysed were synthesised in 2014, 2017 and 2018 using the same method, allowing for a comparison of data between each of the stocks at different stages of storage. As opposed to a majority of literature reports, these agent stocks were not stabilised, nor were they subjected to forced degradation. Using NMR, high resolution (HR) LC-HRMS, GC-(EI)MS and GC-(CI)MS to gain a full insight into the CAS profile, a total of 44 compounds were identified. Of these compounds, 30 were readily identified through accurate mass measurement and NIST library matches. A further seven were identified through extensive LC-HRMS/MS studies, with seven remaining unresolved. Several compounds, identified in minor amounts, were able to be traced back to impurities in the precursor compounds used in the synthesis of VX, and hence may be useful as CAS for source attribution., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)

In this study, we utilized elemental analyser (EA) and gas-chromatography (GC) isotope ratio mass spectrometry (IRMS) and ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) in a comprehensive profiling approach assessing the chromatographic impurity signatures and δ 13 C and δ 15 N isotope ratios of synthetic cannabinoids from police seizures and internet test purchases. Main target of this study was the highly prevalent synthetic cannabinoid MDMB-CHMICA (methyl (2S)-2-([1-(cyclohexylmethyl)-1H-indol-3-yl]formamido)-3,3-dimethylbutaoate). Overall, 61 powder and 118 herbal blend (also called "Spice-Products") samples were analysed using both analytical techniques and evaluated in a joint model to link samples from a common source. As a key finding, three agglomerates of Spice-product samples with similar dates of purchase were identified in the IRMS data, possibly representing larger shipments of MDMB-CHMICA, each produced with the same precursor material, successively delivered to the European market. The three agglomerates were refined into multiple sub-clusters based on the impurity profiling data, each representing an individual synthesis batch. One of the agglomerates identified in the IRMS data was found to consist two groups of four sub-clusters, respectively, with majorly different impurity profiles, demonstrating the necessity for both analytical techniques to extract the maximum amount of information from a limited sample pool. Additionally, 31 samples containing the recently surfaced synthetic cannabinoid Cumyl-PeGaClone (5-pentyl-2-(2-phenylpropan-2-yl)-2,5-dihydro-1H-pyrido[4,3-b]indol-1-one) were analysed for their and δ 13 C and δ 15 N isotope ratios to put the isotopic data recorded for MDMB-CHMNICA in a more global perspective. Three building blocks of precursor chemicals (indole, tert-leucine, cumylamine) potentially used for the synthesis of the two named synthetic cannabinoids were acquired from different global vendors and measured for their δ 13 C and δ 15 N isotope ratios to better understand variations in the isotopic composition of the synthetic cannabinoids and to trace their origin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Two-dimensional liquid chromatography requires orthogonal columns and/or separation principles in the first and second separation dimension. It is sometimes not straightforward to achieve. Chiral columns could expand the toolbox for 2D-LC, but are rarely exploited for this purpose, not least due to missing understanding of retention principles under non-chiral application conditions. To gain more insight, in this study Chiralpak ZWIX(+) and ZWIX(-), based on zwitterionic quinine and quinidine carbamate selectors, were carefully characterized by molecular dynamics simulations, lipophilicity/hydrophilicity measurements of selectors, pH-dependent ζ-potential determinations, and chromatographic characterization in RPLC and HILIC modes combined with unsupervised principal component analysis to extract classification of these columns in comparison to a number of commercial benchmarks (RP, HILIC and mixed-mode columns). The results showed that these chiral columns can be classified as mixed-mode chromatography phases with balanced lipophilic-hydrophilic surface character, excess of negative net charge due to sulfonic acid groups (in spite of weakly basic quinuclidine and quinoline rings), and multimodal applicability (RP, HILIC and polar organic elution modes). Orthogonality mapping in comparison to a number of modern HILIC and mixed-mode columns revealed that Poroshell HILIC-Z (with a zwitterionic ligand on 2.7 μm core-shell particles) can be beneficially combined as second dimension with the ZWIX column for comprehensive LC × LC. The online hyphenation of this 2D-LC system with complementary detection modalities including UV (DAD for chromophoric substances), charged aerosol detection (for universal detection and calibration of non-volatile analytes) and high-resolution mass spectrometry (ESI-QTOF-MS/MS for identification) provided an advanced method for comprehensive impurity profiling, applicable for instance for amino acid pharmaceutical products., (Copyright © 2019 Elsevier B.V. All rights reserved.)