Potential Genotoxic Impurities Research Articles

Development and Validation of HPLC Method for Determination of Trace Level Potential Genotoxic Hydroperoxide Impurity in Canagliflozin

Development and Validation of HPLC Method for Determination of Trace Level Potential Genotoxic Hydroperoxide Impurity in Canagliflozin

Back Cover: Application of simple and sensitive LC‐MS/MS approach for trace level quantification of potential genotoxic impurities in lamivudine salicylate formulations

DOI: 10.1002/sscp.201900032 The cover picture shows the schematic pictorial representation of lamivudine salicylate stepwise for the development and validation for genotoxic impurities. A new LC-MS/MS method developed and validated for the quantification of l-menthol glyoxylate and (1R,2S,5R)-menthyl-5(R,S)-acetoxy-(1,3)-oxathiolane-2(R)-carboxylate genotoxic impurities present in lamivudine salicylate. The genotoxic impuritieswere quantified up to trace level concentration of 0.3 ppm (LOQ) in short run time. The method is highly recommended for regular routine analysis for samples in production stage and to test the stability of lamivudine salicylatedrug dosage.

Application of simple and sensitive LC‐MS/MS approach for trace level quantification of potential genotoxic impurities in lamivudine salicylate formulations

AbstractA new liquid chromatography with tandem mass spectrometry systematic procedure was envisaged for determination of l‐menthol glyoxylate and (1R,2S,5R)‐menthyl‐5(R,S)‐acetoxy‐(1,3)‐oxathiolane‐2(R)‐carboxylate, the potential genotoxic impurities in lamivudine salicylate drug formulation. The process describes an effective use of Hypersil BDS C8 (50 mm × 4.6 mm, 3.0 µm) column retained at 40°C and positive ion electrospray ionization in multiple reaction‐monitoring mode for quantification of two genotoxic impurities. The mobile phase comprises of 60:40 ratio of 5 mM ammonium formate and combination of acetonitrile/methanol (50:50 v/v) pumped at the flow rate of 0.5 mL/min with run time 10 min. This approach was validated for robustness, reproducibility, precision, linearity, accuracy, limit of detection and limit of quantification by following ICH guidelines. The established approach is able to assess two genotoxic impurities at 0.3 ppm (LOQ) against to 2.0 mg/mL of lamivudine. The linearity of this approach is found to 0.3–10.0 ppm, which meets the quantified accepted level (5.0 ppm) in LOQ‐200% range with the correlation coefficients of 0.999 and 0.997. Accuracy was arrived between 98.7–103.1%. The validated method could be highly useful as a good quality assessment tool for quantitation of genotoxic impurities in lamivudine formulation.

An Orthogonal Approach for Determination of Acetamide Content in Pharmaceutical Drug Substance and Base-Contaminated Acetonitrile by GC and GC-MS External Method.

Acetamide is a potential genotoxic impurity; it should control in drug substance based on daily dosage level. It forms from base-contaminated acetonitrile and by-product of some drug substances. The available methods for acetamide in drug substance and water samples were determined by GC-MS using internal standard with critical procedures. These developed and validated methods can assist in evaluating the reaction between acetonitrile and different bases and also determine trace level acetamide in drug substances. The method development was initiated with DB-624, 30 m, 0.32 width and 1.0-μm column. The column was used to validate at the 600ppm TTC value. Similarly, the CP-SIL 5CB, 60 m, 0.32 width, the 5-μm column was used for the remaining TTC values. The validation study was performed for all TTC limits. The % RSD for precision at 600, 60, 20, 10 and 2.5ppm was <15%. The % recovery at all TTC level was in between the 70 and 130%. Solution stability study was performed up to the 24h. At 2.5ppm, the results were <15% variation from the initial value. The linearities from the 50 to 150% concerning TTC values were more than limit of 0.98 correlation coefficient. The limit of detection and limit of quantitation values were 0.4 to the 1.3ppm, respectively, for 2.5ppm TTC limit method.

Development of an Analytical Method for Identification of the Genotoxic Impurity of Quetiapine Fumarate by High-Performance Thin-Layer Chromatography

Genetic mutations, chromosomal breaks, and chromosomal rearrangements, which are induced due to organic impurities, are considered as potential genotoxic impurities. The European Medicines Agency (EMA) and the United States Food and Drug Administration (US FDA) have set a threshold of toxicological concern (TTC) of 1.5 µg per person per day for each impurity. A sensitive and simple high-performance thin-layer chromatography (HPTLC) method has been developed and validated for determination of the potential genotoxic impurity, namely, 2-chloroaniline, at trace levels in quetiapine fumarate. The method was found to be specific and selective for the application. The limit of detection (LOD) and limit of quantification (LOQ) for quetiapine fumarate were found to be 1.27 and 3.87 ng per band. The LOD and LOQ values for 2-chloroaniline were found 0.018 and 0.054 ng per band, respectively. The calibration curve for 2-chloroaniline was linear over a concentration range from 2.5 to 12.5 ng. The method was found to be specific, precise, linear, and accurate and can be employed for monitoring and estimation of levels of 2-chloroaniline in quetiapine fumarate.

Determination of trace methanesulfonates in drug matrix using derivatization and headspace single drop microextraction followed by high-performance liquid chromatography with ultraviolet detection

The selective and sensitive determination of potential genotoxic methanesulfonate impurities in drug substances is highly challenging. A new method is reported for testing of methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and isopropyl methanesulfonate (IPMS) in active pharmaceutical ingredients (APIs). Headspace single drop microextraction (HS-SDME) with room-temperature ionic liquid (RTIL) as extractant was employed to preconcentrate analytes and eliminate the drug matrix simultaneously. In order to increase volatilities for HS extraction and to improve their reactivity of the further derivatization at the same time, sodium iodide (NaI) was added to the sample to derivatize methanesulfonates to the corresponding iodoalkanes. The iodoalkanes in the extract were derivatized with N, N-diethyldithiocarbamate (DDTC) after HS-SDME, followed by separation and detection with high-performance liquid chromatography with ultraviolet detection (HPLC-UV). Several important parameters, including reaction temperature, reaction time and concentration of NaI, sample volume, microdrop volume, stirring rate, salt addition, extraction time, concentration, reaction time and reaction temperature of DDTC were investigated. Under the optimal conditions, LODs and LOQs of all methanesulfonates were 15 ng mL−1 and 40 ng mL−1, respectively. Linearity (correlation coefficient values r > 0.999) and precision (the relative standard deviations were 1.0–4.6%) of six repeated injections were obtained. The recoveries at three spiked concentration levels were all in the range of 86.2–107.5% with the relative standard deviations <3.5%. The method reported here avoids interference of drug substances efficiently and detects methanesulfonate impurities in high sensitivity by HPLC-UV in Imatinib Mesylate and Levofloxacin Mesylate.

Quantification of potential genotoxic impurity in sacubitril/valsartan drug substance at ppm level by LC-MS/MS

Abstract The aim of the present research work is to provoke a validated, sensitive and highly selective LC-MS/MS method for the determination of potential genotoxic impurity namely Sacubitril Ene Ester (SEE) impurity in sacubitril/valsartan drug substance at ppm level. The LC-MS/MS method was developed on waters X-Bridge Phenyl (150mm x 4.6 mm) 3.5 µm in gradient elution at 30°C. Mobile phase-A was 10mM ammonium acetate and mobile phase-B was acetonitrile: methanol: water (60:30:10, %v/v/v) at a constant flow rate and injection volumes are of 0.8 mL/min and 10µL respectively with 30 minutes gradient run time monitored by mass spectrophotometer. Validation was performed on basis of the standard guideline “International Conference on Harmonization” (ICH) guideline Q2(R1). LC-MS/MS is able to quantitate up to 1.1ppm of SEE impurity. Under these LC and MS conditions, SEE impurity was quantified by multiple reaction monitoring pair (most stable ions) m/z of 408.1/262.2. This method has been used for testing and quantitative determination of SEE impurity in sacibitril/valsartan samples at µg levels. The developed LC-MS/MS method was validated and the resulting data found to show the specificity, linearity, accuracy and precision of the proposed methodology. Further the results show that the developed LC-MS/MS method was well suited to quantify the SEE potential genotoxic impurity.

A new HPLC-UV derivatization approach for the determination of potential genotoxic benzyl halides in drug substances.

Benzyl halides, widely used as alkylation reagents in drug synthesis, are potential genotoxic impurities (PGTIs) required to be controlled at trace levels. However, the existing analytical methods for benzyl halides often suffer from matrix interferences or low derivatization efficiency of benzyl chlorides. In this paper, a simple derivatization HPLC-UV method was developed for the analysis of these residual trace benzyl halides in drug substances. 1-(4-Nitrophenyl) piperazine (4-NPP) was selected as a new derivatization reagent because it shifted well the benzyl halides derivatives away to the near visible range (392 nm), which could minimize the matrix interferences from the drug substances and related impurities. Meanwhile, potassium iodide (KI) was used to convert the mixed benzyl halides into benzyl iodides before derivatization. The derivatization parameters were also optimized using the design of experiments (DoE) for achieving the best reaction efficiency. The results showed that the new approach had high specificity and sensitivity, and the LOQs were 7–9 μg g−1 relative to 5 mg mL−1 antipyrine and 17.5–22.5 μg g−1 relative to 2 mg mL−1 oroxylin A. The method is a valuable alternative for the determination of residual benzyl halides in the drug substances.

Identification, isolation, characterization, and UHPLC quantification of potential genotoxic impurities in linagliptin.

During the stress testing of linagliptin, one unknown degradation product (impurity I) on acidic conditions was detected by using high-performance liquid chromatography and subsequently isolated, identified, and characterized by the spectral data (MS, MS/MS, 1D and 2D NMR spectroscopy, and IR spectroscopy) and finally subjected for mechanism analysis. The degradation product (impurity I) and another process-related impurity (impurity II) of linagliptin were found to contain the structural alerts of N-acylated aminoaryl and alkyl halide, respectively, which were both potential genotoxic substances. Hence, a rapid and facile ultra high performance liquid chromatography method was developed for the simultaneous determination of these two potential genotoxic impurities at ppm levels in linagliptin. The factors involved in the method development were discussed and this method was fully validated as per International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines, which proved the method sensitive, selective, and robust. The presented method has been successfully applied to the analysis of real samples from multiple batches. This study will help to risk management of possible genotoxic impurities present in linagliptin.

A simple, sensitive, and straightforward LC–MS approach for rapid analysis of three potential genotoxic impurities in rabeprazole formulations

In the present study, a sensitive and fully validated liquid chromatography with mass spectrometry method was developed for the quantification of three potential genotoxic impurities in rabeprazole drug substance. The separation was achieved on Symmetry C18 column (100×4.6mm, 3.5μm) using 0.1% formic acid in water as mobile phase A and acetonitrile as mobile phase B in gradient elution mode at 0.5mL/min flow rate. Triple quadrupole mass detection with electrospray ionization was operated in selected ion recording mode for the quantification of impurities. The calibration curves were demonstrated good linearity over the concentration range of 1.0-4.5ppm for O-phenylenediamine, 1.8-4.5ppm for 4-nitrolutidine-N-oxide and 1.0-4.5ppm for benzyltriethylammonium chloride with respect to 10mg/mL of rabeprazole. The correlation coefficient obtained in each case was >0.998. The recoveries were found satisfactory over the range between 94.22 and 106.84% for all selected impurities. The method validation was carried out following International Conference on Harmonization guidelines, from which the developed method was able to quantitate the impurities at 1.0ppm for O-phenylenediamine, 1.8ppm for 4-nitrolutidine-N-oxide and 1.0ppm for benzyltriethylammonium chloride. Furthermore, the proposed method was successfully evaluated for the determination of selected impurities from bulk drug and formulation samples of rabeprazole within the acceptable limits.

DETERMINATION OF RESIDUAL DIMETHYL SULFATE IN METHOXSALEN DRUG SUBSTANCE BY PRE-COLUMN DERIVATIZATION WITH STATIC HEADSPACE GAS CHROMATOGRAPHY

Objective: Dimethyl sulfate has been highlighted as potential genotoxic and carcinogenic impurity. A sensitive Headspace gas chromatography (HS-GC) method with pre-column derivatization was developed and validated for the determination of dimethyl sulfate impurity in methoxsalen active pharmaceutical ingredient.Methods: HS-GC method on the column Agilent DB-5, 30m X 0.53 mm, film thickness 1.5 µm, with flame ionization detector (FID) was used. Derivatization reagent concentration, time of reaction and pH of the solution were optimized during method development. This analytical method was evaluated by performing method validation as per ICH guideline.Results: The proposed method was specific, linear, accurate, rugged and precise. The calibration curves showed good linearity over the concentration range of 0.5 μg/ml to 3.0 μg/ml and the correlation coefficient was 0.999. Method had very low limit of detection (LOD) and limit of quantification (LOQ) 2.0 μg/g and 5.0 μg/g respectively. Accuracy was observed within 98.1%–104.5%.Conclusion: The developed method was demonstrated to be accurate, robust and sensitive for the determination of dimethyl sulfate impurity in methoxsalen drug substance.

A NOVEL HPLC METHOD FOR ESTIMATION OF GENOTOXIC IMPURITY 3-ACETAMIDOBENZENE 1, 2 DICARBOXYLIC ACID IN KEY STARTING MATERIAL 3-ACETAMIDOPTHALIC ANHYDRIDE OF APREMILAST API

3-Acetamidobenzene -1, 2-dicarboxylic acid is a potential genotoxic impurity which gets formed during synthesis of 3- acetamidopthalic anhydride, a Key Starting Material (KSM) for manufacturing of apremilast API. During handling upon exposure to moisture, the anhydride ring of KSM 3-acetamidopthalic anhydride opens to form the acid. Hence Reverse phase HPLC method is not a feasible and robust option for estimation of this impurity. To overcome these problems a normal phase HPLC method is developed and proposed in this research work. Immobilized Chiral pack IA column from Daicel is used for estimation. n-Hexane and isopropyl alcohol in 90:10 (v/v) ratio modified with 0.1% trifluroacetic acid is used as mobile phase. Method is validated as per ICH guidelines. Limit of Detection (LOD) and Limit of Quantification (LOQ) are found to be 0.47 ppm (0.0047%) and 1.42 ppm (0.0142%), respectively. The method is linear over LOQ to 150%. Recovery is within limits (80-120%). Method is robust for parameters like mobile phase composition, flow rate, wavelength changes and column oven temperature.

A Selective and Sensitive Method Development and Validation by LC-MS/MS Approach for Trace Level Quantification of Two Potential Genotoxic Impurities in Albendazole Drug Substance

A Selective and Sensitive Method Development and Validation by LC-MS/MS Approach for Trace Level Quantification of Two Potential Genotoxic Impurities in Albendazole Drug Substance

Determination of residual 4-nitrobenzaldehyde in chloramphenicol and its pharmaceutical formulation by HPLC with UV/Vis detection after derivatization with 3-nitrophenylhydrazine

4-Nitrobenzaldehyde is the synthetic raw material and an important photodegradation product of chloramphenicol. With a structural “alert” of human genotoxic potential and reported mutagenicity, this compound should be controlled in drug substances as a potential genotoxic impurity. However, current analysis methods require complex pre-treatment processes and/or lack sufficient specificity and sensitivity. Nitrophenylhydrazine is a common carbonyl derivatization reagent used to determine the residual aromatic aldehydes in drug samples. In the present study, we report an unexpected advantage of 3-nitrophenylhydrazine hydrochloride as a derivatization reagent in the derivatization high-performance liquid chromatography-ultraviolet detection method to determine 4-nitrobenzaldehyde in chloramphenicol samples. Compared with other nitro-substituted phenylhydrazines, 3-nitrophenylhydrazine hydrochloride can minimize drug matrix and derivatization reagent interferences, since the maximum absorption wavelength of its derivative is significantly red-shifted to 397 nm. The derivatization conditions have been optimized in terms of reaction efficiency, including reaction temperature, time, and diluting solvent, through a design of experiments. As a result, after reaction with 500 μg mL−1 of 3-nitrophenylhydrazine hydrochloride in acetonitrile-water (70:30, v/v) at 60 °C for 30 min, the developed HPLC method could be used to determine 4-nitrobenzaldehyde with a limit of detection of 0.009 μg mL−1. The method was then validated and applied for the determination of residual 4-nitrobenzaldehyde in chloramphenicol and its eye-drop samples.

Development and Validation of GCMS method for the Detection and Quantification of Potential Genotoxic Impurity Ethyl 4-bromobutyrate in Tolvaptan Tablets

Development and Validation of GCMS method for the Detection and Quantification of Potential Genotoxic Impurity Ethyl 4-bromobutyrate in Tolvaptan Tablets

Advances on genotoxic impurities of sulfonate esters in pharmaceuticals

This article reviews the refinement of regulatory guidelines and progress of research on the control of genotoxic impurities in pharmaceuticals in the last decade. It outlines advances in the regulatory requirements for genotoxic impurities from strict avoidance to the currently accepted concept of threshold of toxicological concern (TTC), which is based on risk control considerations. Specific control limits, which are required by predominant administrative regulatory agencies, such as U. S. Food and Drug Administration (FDA), European Medicines Agency (EMA) and the international conference on harmonisation of technical requirements for registration of pharmaceuticals for human use (ICH), etc. Sulfonate esters, an important class of potential genotoxic impurities, are usually generated by side-reactions between sulfonic acids or their derivatives and relative low molecular mass alcohols, such as methanol, ethanol, and isopropanol. The resulting sulfonate esters are characterized with diverse chemical structures. Reaction mechanisms of the formation of sulfonate esters and various strategies to control them have been schematically described. A detailed summary has been given for the analytical methodology developed using high performance liquid chromatography (HPLC) and gas chromatography (GC) to determine trace amounts of sulfonate esters in pharmaceuticals. Furthermore, we have comprehensively discussed the options for the chromatographic methods, sample pretreatments, and derivatization methods, as well as each method's sensitivity and recovery at trace level. This review intended to provide constructive suggestions for the rational control of sulfonate esters in pharmaceuticals to ensure their clinical safety.

DETERMINATION OF RESIDUAL METHYL IODIDE IN TEDIZOLID PHOSPHATE AND ALOGLIPTIN BENZOATE BY STATIC HEADSPACE GAS CHROMATOGRAPHY WITH ELECTRON CAPTURE DETECTION

Methyl iodide has been highlighted as a potential genotoxic impurity (PGI). A sensitive HS-GC method with ECD was developed and validated for the determination of methyl iodide impurity in Tedizolid phosphate, an active pharmaceutical ingredient. HS-GC method on DB-624,column 60m X 0.53mm, film thickness 3μm, with electron capture detector (ECD) was used. The proposed method was specific, linear, accurate, rugged and precise. The calibration curves showed good linearity over the concentration range of 0.78 μg/g to 11.7 μg/g and the correlation coefficient was 0.997. Method had very low limit of detection (LOD) and limits of quantification (LOQ) are 0.23 μg/g and 0.78 μg/g, respectively, with respect to test concentration. Accuracy was observed within the range 96.0% to 103.2%. This method is a further extended good quality control tool for low level quantitation of methyl iodide impurity in another API, alogliptin benzoate.

Analysis of potential genotoxic impurities in rabeprazole active pharmaceutical ingredient via Liquid Chromatography-tandem Mass Spectrometry, following quality-by-design principles for method development

A novel Liquid Chromatography-tandem mass spectrometry (LC–MS/MS) method is presented for the quantitative determination of two potential genotoxic impurities (PGIs) in rabeprazole active pharmaceutical ingredient (API). In order to overcome the analytical challenges in the trace analysis of PGIs, a development procedure supported by Quality-by-Design (QbD) principles was evaluated. The efficient separation between rabeprazole and the two PGIs in the shortest analysis time was set as the defined analytical target profile (ATP) and to this purpose utilization of a switching valve allowed the flow to be sent to waste when rabeprazole was eluted. The selected critical quality attributes (CQAs) were the separation criterion s between the critical peak pair and the capacity factor k of the last eluted compound. The effect of the following critical process parameters (CPPs) on the CQAs was studied: %ACN content, the pH and the concentration of the buffer salt in the mobile phase, as well as the stationary phase of the analytical column. D-Optimal design was implemented to set the plan of experiments with UV detector. In order to define the design space, Monte Carlo simulations with 5000 iterations were performed. Acceptance criteria were met for C8 column (50×4mm, 5μm), and the region having probability π≥95% to achieve satisfactory values of all defined CQAs was computed. The working point was selected with the mobile phase consisting ‎of ACN, ammonium formate 11mM at a ratio 31/69v/v with pH=6,8 for the water phase. The LC protocol was transferred to LC–MS/MS and validated according to ICH guidelines.

Synthesis and Quantitation of Genotoxic impurity 5-cyano-2-((4-fluorophenyl) (hydroxy) benzyl 4-methyl benzene sulfonate in Escitalopram Oxalate by RP-UPLC

The objective of the research work is to synthesize potential genotoxic impurity 5-cyano-2-((4-fluorophenyl) (hydroxyl) benzyl 4-methyl benzene sulfonate and to develop suitable UPLC method to quantify the above genotoxicimpurity in Escitalopram Oxalate at 15 ppm level. The above genotoxic impurity was synthesized by regio selective tosyaltion of Diol compound, under controlled temperature conditions at 0-5C with TsCl/pyridine/chloroform and characterized. A new UPLC method was developed by using UPLC BEH Shield RP18 100 x 2.1 mm, 1.7 column. The mobile phase used is the mixture of 0.05% ortho phosphoric acid and acetonitrile in the ratio of 8:2 (v/v) as solvent-A and 3:7 (v/v) as solvent-B at a flow rate of 0.4 mL/minute. Detector wavelength monitored at 228nm and column temperature was maintained at 27C. The UPLC method was validated as per International conference on harmonization guidelines. This method is proven as highly sensitive with a detection limit of 5ppm and quantification limit of 15 ppm. Regression analysis showed that the correlation coefficient value of 0.99999. The accuracy of the method was established based on the recovery obtained for 5-cyano-2-((4-fluorophenyl) (hydroxyl) benzyl 4-methyl benzene sulfonate. The present research work provided the route of synthesis as well as an advanced analytical methodology to quantify the above critical Genotoxic impurity known as 5-cyano-2-((4-fluorophenyl) (hydroxyl) benzyl 4-methyl benzene sulfonate in Escitalopram oxalate.

A SELECTIVE AND SENSITIVE METHOD DEVELOPMENT AND VALIDATION BY LC-MS/MS APPROACH FOR TRACE LEVEL QUANTIFICATION OF POTENTIAL GENOTOXIC IMPURITY OF BOC EPOXIDE IN ATAZANAVIR SULPHATE DRUG SUBSTANCE

Objective: To develop and validate a selective, sensitive, rapid and accurate method using LC-MS/MS technique to achieve efficient separation between active pharmaceutical ingredient (Atazanavir sulphate) and genotoxic impurity (BOC epoxide).Methods: The quantification was carried out using the column puro sphere star RP 18 e (length 150 mm, internal diameter 4.6 mm, particle size 3.0 µm) with electrospray ionization in multiple reaction monitoring (MRM) detection mode. Eluent-A was 0.1% formic acid in water and eluent-B was 0.1% formic acid and 0.1% ammonium hydroxide solution (25%) in acetonitrile. The isocratic mode of elution was carried out for the elution of impurity with the shorter run time of 6 min. The flow rate was 1.0 ml/min and column oven temperature was maintained 25 °C.Results: The method was validated as per ICH guidelines and arrived the limit of detection and limit of quantification for the potential genotoxic impurity and found to be 0.2 ppm and 0.5 ppm. The developed method was found linear in the concentration range of 0.5 ppm to 6 ppm and accuracy results were within the range. Conclusion: The developed short span method found to be selective, sensitive, precise and accurate for the quantification of the BOC epoxide genotoxic impurity in atazanavir sulphate drug substance.