Potential Genotoxic Impurities Research Articles

A novel LC-QTOF-MS/MS method for trace level identification and quantification of potential genotoxic impurity, 5-Nitro-2-(propylthio)pyrimidine-4,6-diol in ticagrelor drug substance

An important objective of the present work is to develop and validate a novel LC-QTOF-MS/MS method to identify and quantify 5-Nitro-2-(propylthio) pyrimidine-4,6-diol genotoxic impurity at trace level (∼8.33 ppm) in ticagrelor drug substance, is an antiplatelet medicine used in the treatment of heart attacks and strokes. The potential genotoxic impurities must be controlled below acceptance limit during process of ticagrelor synthesis and release. In reversed-phase mode, the ACQUITY UPLC CSH C18 (2.1 mm x 100 mm, 1.7 μm) with ambient column temperature was used. 0.1% formic acid in Milli-Q water and methanol in the ratio of 30:70 v/v used as mobile phase A and mobile phase B with isocratic elution mode and the run time was 8.0 min. The flow rate was maintained at 0.25 mL/min, autosampler temperature was 5°C, Injection volume was 20 μL. The Retention time of 5-Nitro-2-(propylthio)pyrimidine-4,6-diol was found at 1.345 minutes. Limit of detection (LOD) and limit of quantification (LOQ) were found at 0.56 ppm and 1.67 ppm, respectively. The 5-Nitro-2-(propylthio)pyrimidine-4,6-diol was linear (R2=0.9986) over the concentration range 1.666 to 12.495 ppm. The method was accurate and precise, it was demonstrated by calculating the %mean recovery values ranging from 98.0-106.9%.

Determination of genotoxic impurities of aromatic aldehydes in pharmaceutical preparations by high performance liquid chromatography after derivatization with N-Cyclohexyl-4-hydrazino-1,8-naphthalenediimide

The detection of aromatic aldehydes, considered potential genotoxic impurities, holds significant importance during drug development and production. Current analytical methods necessitate complex pre-treatment processes and exhibit insufficient specificity and sensitivity. This study presents the utilization of naphthalenediimide as a pre-column derivatisation reagent to detect aromatic aldehyde impurities in pharmaceuticals via high-performance liquid chromatography (HPLC). We screened a series of derivatisation reagents through density functional theory (DFT) and investigated the phenomenon of photoinduced electron transfer (PET) for both the derivatisation reagents and the resulting products. Optimal experimental conditions for derivatisation were achieved at 40 °C for 60 min. This approach has been successfully applied to detect residual aromatic aldehyde genotoxic impurities in various pharmaceutical preparations, including 4-Nitrobenzaldehyde, 2-Nitrobenzaldehyde, 1,4-Benzodioxane-6-aldehyde, and 5-Hydroxymethylfurfural. The pre-column derivatisation method significantly enhanced detection sensitivity and reduced the limit of detection (LOD), which ranged from 0.002 to 0.008 μg/ml for the analytes, with relative standard deviations < 3 %. The correlation coefficient (R2) >0.998 demonstrated high quality. In chloramphenicol eye drops, the concentration of 4-Nitrobenzaldehyde was measured to be 8.6 µg/mL below the specified concentration, with recoveries ranging from 90.0 % to 119.2 %. In comparison to existing methods, our work simplifies the pretreatment process, enhances the sensitivity and specificity of the analysis, and offers comprehensive insights into impurity detection in pharmaceutical preparations.

A Rapid, Selective and Sensitive Electrospray Ionization Assisted LC-MS Method for Ranolazine and Identification of Its Two Potential Genotoxic Impurities

A Rapid, Selective and Sensitive Electrospray Ionization Assisted LC-MS Method for Ranolazine and Identification of Its Two Potential Genotoxic Impurities

DNA adductomics aided rapid screening of genotoxic impurities using nucleosides and 3D bioprinted human liver organoids

Current genotoxicity assessment methods are mainly employed to verify the genotoxic safety of drugs, but do not allow for rapid screening of specific genotoxic impurities (GTIs). In this study, a new approach for the recognition of GTIs has been proposed. It is to expose the complex samples to an in vitro nucleoside incubation model, and then draw complete DNA adduct profiles to infer the structures of potential genotoxic impurities (PGIs). Subsequently, the genotoxicity is confirmed in human by 3D bioprinted human liver organoids. To verify the feasibility of the approach, lansoprazole chloride compound (Lanchlor), a PGI during the synthesis of lansoprazole, was selected as the model drug. After confirming genotoxicity by Comet assay, it was exposed to different models to map and compare the DNA adduct profiles by LC-MS/MS. The results showed Lanchlor could generate diverse DNA adducts, revealing firstly its genotoxicity at molecular mechanism of action. Furthermore, the largest variety and content of DNA adducts were observed in the nucleoside incubation model, while the human liver organoids exhibited similar results with rats. The results showed that the combination of DNA adductomics and 3D bioprinted organoids were useful for the rapid screening of GTIs.

Development and Validation of Simple and Stable LCMS Method for the Quantification of Potential Genotoxic Impurities in Ozenoxacin Pure Drug and its Commercial Preparations

Ozenoxacin is an antibiotic drug prescribed to treat various skin infections caused by various bacteria. Various chemical mechanisms such as stille coupling, Buchwald–Hartwig coupling, cyclization and saponification are involved during the process of synthesis of Ozenoxacin. In the process of synthesis, there is a possibility of the formation of related impurities and among them, some are genotoxic impurities. To date, in literature, there is no method reported for analysing Potential Genotoxic Impurities (PGIs) in Ozenoxacin and hence this study was initiated to develop an LCMS method for quantification of two genotoxic impurities of Ozenoxacin viz., nitroso impurity, ester impurity. The analytes were resolved on Alltima C18 column (150×4.6mm; 5 μm particle size) using 0.01 mM ammonium acetate at pH 4.8 and methanol in 80:20 (v/v) at 0.5 mL/min flow rate and 10 μ sample injection volume. The multiple reaction monitoring of the mass fragments confirms the parent ion at m/z of 364, 393 and 496 for Ozenoxacin, Nitroso and Ester impurity respectively with characteristic product ion at m/z 196. The method has a linearity range of 0.05 μg/mL to 1.0 μg/mL for three analytes with detection limits of 0.015, 0.011 and 0.015 μg/mL for Ester impurity, Ozenoxacin and Nitroso impurity respectively. The method was validated and produces acceptable results and can successfully separate the potential genotoxic impurities in spiked commercial samples. Based on the findings, it was concluded that this method can be practically useful for the identification and quantification of potential genotoxic impurities and may apply to the safe use of Ozenoxacin in clinical treatment.

Identification, trace level quantification, and in silico assessment of potential genotoxic impurity in Famotidine

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.

In silico Toxicity Assessment and Quantification of Potential Genotoxic Impurity in Olmesartan Medoxomil Drug Substance using GC-MS/MS

This study was designed to assess the in silico toxicity of 4-chloromethyl-5-methyl-1,3-dioxol-2-one (4-CMMD) in olmesartan medoxomil (OLM) using sophisticated advanced analytical GC-MS/MS method. The developed GC-MS/MS method is more sensitive as well as selective, for trace level analysis of genotoxic impurity 4-chloromethyl-5-methyl-1,3-dioxol-2-one (4-CMMD) in olmesartan medoxomil (OLM). In silico genotoxicity of 4-CMMD have been confimed by ICH M7 guidelines and tested to be “POSITIVE” in both knowledge and statistical based approaches. The conditions of gas chromatographic separation and mass spectrometry were optimized on stationary phase DB-35MS, helium carrier gas as at a flow rate of 1.5 mL/min. Quantification was performed in multiple reaction monitoring (MRM) mode. The absence of interference at the retention time of 4-CMMD indicates that the newly developed approach is very specific and selective for accurately measuring trace levels of impurities. Additionally, this method provided linear results which were validated by linearity solutions, with concentrations ranging from 3.74 to 45.12 ppm and an observed coefficient of regression of 0.9981. Sensitivity results shows this method is more sensitive detection limit (DL) achieved at 1.23 ppm and quantification limit (QL) achieved at 3.74 ppm. The developed method is precise and accurate according to the precision results, which show RSD values &lt; 10% and recovery &gt; 90%, both of which are within acceptable standards. The solution stability of the samples was assessed at both room temperature and refrigerated settings, and it was found to be stable for a period of 48 h. As a result, this method has been employed for the intented purpose of quantifying 4-CMMD at the trace levels in testing laboratories, pharmaceutical analytical laboratories, and quality control laboratories.

Analytical determination of ethylenediamine impurity in tripelennamine hydrochloride by gas chromatography-mass spectrometry using phthalaldehyde as the derivatizing agent.

In the pharmaceutical industry, effective risk management and control strategies for potential genotoxic impurities are of paramount importance. The current study utilized GC-MS to evaluate a precise, linear, and accurate analytical method for quantifying ethylenediamine present in tripelennamine hydrochloride using phthalaldehyde as a derivatizing agent. When phthalaldehyde is sonicated for 10 min at room temperature, it reacts with ethylenediamine to form (1z,5z)-3,4-dihydrobenzo[f][1,4]diazocine. This approach minimizes matrix interference issues and resolves sample preparation difficulties encountered during ethylenediamine identification in GC-MS. In this method, helium serves as the carrier gas, while methanol acts as the diluent. The stationary phase consists of a DB-5MS column (30 m × 0.25 mm × 0.25 μm) with a flow rate of 1.5 mL/min. The retention time of (1z,5z)-3,4-dihydrobenzo[f][1,4]diazocine was determined to be 6.215 min. The method validation demonstrated limits of detection and quantification for (1z,5z)-3,4-dihydrobenzo[f][1,4]diazocine at 0.4 and 1.0 ppm, respectively, with a linearity range spanning from 1 to 30 ppm concentration with respect to the specification level. System suitability, precision, linearity, and accuracy of the current method were assessed in accordance with guidelines, yielding results deemed suitable for the intended use.

Quantitative determination of potential genotoxic impurities in metformin hydrochloride and empagliflozin tablets through ultra-performance liquid chromatography[sbnd]mass spectrometry

An increasing number of drug research institutions consider genotoxic impurity research a core task in drug research and development. Peroxides in drugs may directly or indirectly attack and damage cell DNA, posing a potential carcinogenic risk to the human body. Currently, the literature only studies hydroperoxide impurities, and benzyl peroxide has not been studied yet. In this study, an effective method for ultra-performance liquid chromatographymass spectrometry (UPLCMS/MS) was established and verified to detect and quantify the potential genotoxic impurity (PGI), empagliflozin benzyl peroxide, in metformin–empagliflozin combination formulations, which has not been reported thus far. A Waters ACQUITY UPLC HSS T3 (3.0 ×100 mm, 1.8 µm) column was used to achieve chromatographic separation with gradient elution. The mass spectrometry conditions were optimized using electrospray ionization in the negative mode. Following the International Conference of Harmonization (ICH) guidelines, this methodology can quantify PGIs at 1.35 ng/mL (5.4 ppm) in samples. This validated method exhibited good linearity over a concentration range of 5.4 to 36 ppm, and the accuracy of this method was in the range of 83.2–95.0% for empagliflozin benzyl peroxide. This approach fills the gap in the detection method for benzyl peroxide impurities in metformin hydrochloride and empagliflozin tablets, providing technical support for the quality control of the drug.

Optimization of LC-MS/MS Analytical Method for Trace Level Quantification of Potential Genotoxic Impurities in Siponimod Pure Drug and Formulations

Optimization of LC-MS/MS Analytical Method for Trace Level Quantification of Potential Genotoxic Impurities in Siponimod Pure Drug and Formulations

Simultaneous and trace-level quantification of four benzene sulfonate potential genotoxic impurities in doxofylline active pharmaceutical ingredients and tablets using high-performance liquid chromatography with ultraviolet detection.

In the production of doxofylline, the common occurrence of toxic p-toluene sulfonate generation prompted the development and validation of a method using HPLC with ultraviolet detection (HPLC-UV). This method is designed for detecting four potential genotoxic impurities (PGIs) present in both doxofylline drug substance and tablets, with a focus on the UV-absorbing group p-toluene sulfonate. The four impurities were methyl 4-methylbenzenesulfonate (PGI-1), ethyl 4-methylbenzenesulfonate (PGI-2), 2-hydroxyethyl 4-methylbenzenesulfonate (PGI-3), and 2-(4-methylphenyl)sulfonyloxyethyl 4-methylbenzenesulfonate (PGI-4). In this method, chromatographic separation was achieved using a Waters Symmetry C18 column (250 mm × 4.6 mm, 5 μm). The mobile phases consisted of 20% acetonitrile as mobile phase A and pure acetonitrile as mobile phase B, operating in gradient elution mode at a flow rate of 1.0 mL/min. According to the guidelines of the International Conference on Harmonization, it was determined that this method could quantify four PGIs at 0.0225 μg/mL in samples containing 60 mg/mL. The validated approach demonstrated excellent linearity (R2 > 0.999) across the concentration range of 30%-200% (relative to 0.075 μg/mL doxofylline) for the four PGIs. The accuracy of this method for the four PGIs ranged from 94.8% to 100.4%. The reverse-phase-HPLC-UV analytical method developed in this study is characterized by its speed and precision, making it suitable for the sensitive analysis of benzene sulfonate PGIs in doxofylline drug substances and tablets.

Investigation of aclidinium bromide degradation by stability-indicating HPLC methods, characterization of impurities by NMR, and identification of degradation products by LC-MS

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 1H and 13C 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.

Stability‐indicating method for quantification of potential genotoxic impurity and other organic impurities of chlorzoxazone using hyphenated techniques

AbstractA simple, reliable, and stability‐indicating RP‐HPLC‐UV method was developed and validated for the estimation of related substances (p‐chlorophenol and unknown impurities) of chlorzoxazone (skeletal muscle relaxant). Along with this as the compound contains a potential genotoxic impurity (2‐amino‐4‐chlorophenol), which needs to have more sensitivity in quantification, we have also developed and validated a separate RP‐HPLC‐mass spectrometry (MS)/MS method for the estimation of 2‐amino‐4‐chlorophenol impurity. Both methods (RP‐HPLC‐UV &amp; RP‐HPLC–MS/MS) were validated in accordance with International Council for Harmonization Q2(R1) and were found to be precise, accurate, robust, linear, and specific with limit of quantification values established with respect to 100% of test concentration, 0.018% w/w of p‐chlorophenol by RP‐HPLC‐UV, and 2 ppm of 2‐amino‐4‐chlorophenol by RP‐HPLC–MS.

Development and Validation of Headspace-Gas Chromatography (HS-GC) Method for the Determination of Potential Genotoxic Impurities in Meloxicam

The development and validation of a specific Headspace-Gas Chromatography (HS-GC) method for the determination of genotoxic impurities (isopropyl chloride, propionaldehyde, 2-chloropropanol, and chloromethane) in Meloxicam. The chromatographic separation of these impurities was accomplished using a nitrogen carrier gas on a DB-624 (6% cyanopropyl phenyl + 94% dimethyl polysiloxane fused silica) (75 m x 0.53 mm, 3.0 µ) GC analytical column. The Flame Ionization Detector (FID) served as the detector with optimized temperatures set at 260°C for the detector and 240°C for the injector port. Additionally, an injection volume of 1.0 mL with a split ratio mode was employed. The performance of this method was assessed through parameters, including system suitability, specificity, linearity, range, accuracy, limit of detection (LOD), limit of quantification (LOQ) and precision. The limit of detection and limit of quantification for isopropyl chloride, propionaldehyde, 2-chloropropanol, and chloromethane were determined to be 2 and 4 ppm, respectively, and for chloromethane 0.8 and 1.6 respectively. The correlation coefficient of the linearity experiment was 0.999 for propionaldehyde, 2-chloropropanol, chloromethane and 1 for isopropyl chloride compounds. Furthermore, the accuracy assessment involved spiking the genotoxic impurities at various levels, demonstrating favourable recovery percentages well within the acceptable range of 80–120% of the limit level. These compelling results substantiate the suitability of the proposed HS-GC method for the accurate determination of isopropyl chloride, propionaldehyde, 2-chloropropanol, and chloromethane in Meloxicam.

In-silico Toxicity Assessment and Trace Level Quantification of Veratryl Chloride a Potential Genotoxic Impurity in Ivabradine Hydrochloride using LC-MS/MS

A sensitive and selective liquid chromatography-mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) mode was developed and validated for trace analysis of 4-(chloromethyl)-1, 2-dimethoxybenzene (Veratryl chloride) a potential genotoxic impurity in Ivabradine hydrochloride (IVB). Chromatographic separation was performed on a Poroshell 120EC C18 (50 × 3.0 mm, 2.7 μm) column using a mixture of 10 mM ammonium formate and acetonitrile in isocratic elution mode at a 0.25 mL/min flow rate. A simple pre-column derivatization with di-ethylamine was employed for the derivatization of the veratryl chloride. The developed LC-MS/MS method was linear and accurate in the 1.5–10.0 ppm concentration range with r2 ˃ 0.999 and percent recoveries greater than 90%. The developed method was precise with RSD (%) of not more than 4.5%. In-silico genotoxicity and carcinogenicity potential of veratryl chloride was assessed using ICH M7 principles found to be positive. The developed method can identify and quantify veratryl chloride in IVB, hence can be applied by quality control labs of pharmaceutical industries for trace quantification.

Identification and Determination of Impurities in a New Therapeutic Agent for Fatty Liver Disease.

Methyl 7,7'-dimethoxy-5'-(morpholinomethyl)-[4,4'-bibenzo[d][1,3] dioxole]-5-carboxylate methanesulfonate (IMM) is an innovative drug for the treatment of nonalcoholic fatty liver disease (NAFLD) owing to its high efficacy and low toxicity. In this study, five minor impurities (I, II, III, IV, and V) were identified and analyzed using spectroscopic evidence, chemical synthetic methods, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The impurities included hydrolysates and oxidation by-products extracted from both the drug in its final formulation and during synthesis. Toxicity prediction revealed potential carcinogenicity of impurity V containing an N-oxygen fragment. A reliable and selective HPLC method for the quantitative analysis of impurities I-IV and a sensitive HPLC-MS/MS method for potential genotoxic impurity V were developed and optimized. The methods were validated based on the International Council for Harmonization guidelines. Satisfactory linearity was obtained for the analytes over the range of 0.1-2.0 μg/mL for impurities I-IV and 0.3-30.0 ng/mL for impurity V, and in all cases, the fitting correlation coefficients exceeded 0.999. The obtained limits of detection values were 0.05 ng/mL and 0.005 μg/mL for impurity V and impurities I-IV, respectively. The precision and repeatability of the methods were less than 1.08% and 8.72% for each impurity. The recovery percentages of all impurities were in the range of 91.18%-111.27%, with the relative standard deviation of less than 3.69%. The greenness assessment of the HPLC method and the HPLC-MS/MS method were evaluated by using AGREE software with a score value of 0.72 and 0.68, respectively. The recommended procedures that were accurate, specific, and ecofriendly were applied to the existing active pharmaceutical ingredients of IMM, and they generated satisfactory results.

Determination of Two Potential Genotoxic Impurities in Crisaborole API by UPLC-MS/MS

Background: Atopic dermatitis (AD) is an allergic skin disease that can be inherited. Crisaborole is a specific drug developed to deal with this disease. The compounds of 4-(4-Bromo-3-formyl-phenoxy)-benzonitrile and 4-(4-(4-bromo-3-formylphenoxy)-3- formylphenoxy)benzonitrile are the representative impurities in the synthesis of Crisaborole active pharmaceutical in gradient (API). Owing to the genotoxic impurities that could lead to cancer and gene mutations, it is necessary to develop a simple, efficient, sensitive, and accurate method to detect these impurities. In this study, the 4-(4-Bromo-3-formyl-phenoxy)-benzonitrile and 4-(4-(4-bromo-3-formylphenoxy)-3-formylphenoxy)benzo- nitrile present in Crisaborole API were detected using UPLC-MS/MS. Methods: The separation was made on a ZORBAX Eclipse XDB-Pheny column (4.6 mm ×75mm, 3.5 μm) with the mobile phase of acetonitrile solution containing 0.1% trifluoroacetic acid (A) and water containing 0.1% formic acid (B) in gradient elution mode. Quantification was performed using positive ion electrospray ionization (ESI), and the contents of two compounds were determined using the multiple reaction monitoring (MRM) modes. Results: The quantitative analytical method was fully validated with respect to linearity (r&gt;0.9998), sensitivity, precision, accuracy (the average recovery of two impurities was 84.1% to 90.7%), and robustness. Conclusion: Three batches of samples were detected by UPLC-MS/MS, indicating that the proposed approach was applicable for the quality evaluation of Crisaborole.

A sensitive UPLC-MS/MS method for the simultaneous assay and trace level genotoxic impurities quantification of SARS-CoV-2 inhibitor-Molnupiravir in its pure and formulation dosage forms using fractional factorial design

Two potential genotoxic impurities were identified (PGTIs)-viz. 4-amino-1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (PGTI-1), and 1-(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2,4(1H,3H)-one (PGTI-II) in the Molnupiravir (MOPR) synthetic routes. COVID-19 disease was treated with MOPR when mild to moderate symptoms occurred. Two (Q)-SAR methods were used to assess the genotoxicity, and projected results were positive and categorized into Class-3 for both PGTIs. A simple, accurate and highly sensitive ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was optimized for the simultaneous quantification of the assay, and these impurities in MOPR drug substance and formulation dosage form. The multiple reaction monitoring (MRM) technique was utilized for the quantification. Prior to the validation study, the UPLC-MS method conditions were optimised using fractional factorial design (FrFD). The optimized Critical Method Parameters (CMPs) include the percentage of Acetonitrile in MP B, Concentration of Formic acid in MP A, Cone Voltage, Capillary Voltage, Collision gas flow and Desolvation temperature were determined from the numerical optimization to be 12.50 %, 0.13 %, 13.6 V, 2.6 kV, 850 L/hr and 375 °C, respectively. The optimized chromatographic separation achieved on Waters Acquity HSS T3 C18 column (100 mm × 2.1 mm, 1.8 µm) in a gradient elution mode with 0.13% formic acid in water and acetonitrile as mobile phases, column temperature kept at 35 °C and flow rate at 0.5 mL/min. The method was successfully validated as per ICH guidelines, and demonstrated excellent linearity over the concentration range of 0.5–10 ppm for both PGTIs. The Pearson correlation coefficient of each impurity and MOPR was found to be higher than 0.999, and the recoveries were in between the range of 94.62 to 104.05% for both PGTIs and 99.10 to 100.25% for MOPR. It is also feasible to utilise this rapid method to quantify MOPR accurately in biological samples.

GC–MS method for the quantification of potential genotoxic thionyl chloride impurity in Teneligliptin drug substance

The compounds with poor volatility and thermal stability, and injector interaction can end up with non-reproducible peaks and shapes. Using the GC–MS technique, this research succeeded with the conversion of potentially genotoxic thionyl chloride into diethyl sulfite by treating it with ethanol. The derivatization procedure was optimized with excellent accuracy and precision on the silica column (coated with 6% cyanopropylphenyl and 94% polydimethylsiloxane ZB-624, 60 m × 0.25 mm × 1.4 μm) through selective ion monitoring (SIM) mode. The detection and quantification limits of thionyl chloride (as diethyl sulfite) were 0.50 and 1.50 ppm, respectively. The method was linear (1.5 to 56 ppm) with a correlation coefficient of 0.998 and the average drug substance recovery was 108 %. The established method was verified as linear, precise, and specific, and applicable for the low-level detection and quantification of thionyl chloride in Teneligliptin and other gliptins.

UHPLC-MS/MS Method Development and Validation for the Genotoxic Impurities Trimethyl Phosphate and Triisopropyl Phosphate of Elagolix Sodium.

Elagolix sodium is a gonadotropin-releasing hormone (GnRH) receptor antagonist that inhibits endogenous GnRH signaling by competitively binding to GnRH receptors in the pituitary gland to treat moderate to severe pain associated with endometriosis. To keep the safety and quality of the drug, a fast quantitative method by reversed-phase ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry has been developed and validated for the identification, assay and estimation of potential genotoxic impurities trimethyl phosphate and triisopropyl phosphate in commercial batches of this active pharmaceutical ingredient in accordance with International Conference on Harmonization guidelines Q2 and M7. The method was validated by assessing specificity, sensitivity, linearity, the limit of quantification and detection, accuracy, precision and robustness for above analytes at a very low concentration, whose quantification and detection limits reached to 24 and 4.8pg/ml, respectively, and the total run time for a single injection was 6min.