Potential Genotoxic Impurities Research Articles

A highly sensitive and selective method for the development and validation of potential genotoxic impurity in Raltegravir by high-throughput UPLC-MS/MS

Raltegravir is a human immunodeficiency virus (HIV) integrase inhibitor, a novel anti-AIDS drug. To quantify genotoxic impurities (GTIs), conventional analysis techniques are inadequate as a result, there is a high need to develop critical analytical methods for drug analysis using the hyphenated analytical technique. A highly sensitive and selective method was developed and validated for the estimation of genotoxic impurity in Raltegravir Active pharmaceutical ingredient (API) by using UPLC-MS/MS. The UPLC-MS/MS analysis for the quantification of GTI was performed on the symmetry C18 (150 mm×4.6 mm,3.5μm) analysis column and separation of genotoxic impurity was achieved under a gradient mode system using a mobile phase comprising 1% acetic acid as mobile phase (A) and acetonitrile as mobile phase (B) with a column temperature of 40°C and flow rate of 0.5 mL/min. A triple mass quadrupole detector was used to measure GTI by combining with the negative-electrospray ionization used in multi-response monitoring mode (MRM). The newly developed method was validated as per ICH guidelines and was able to measure GTI at 2.0 ppm for about 10mg/mL solution of Raltegravir. The %RSD obtained for GTI for six preparations was 1.74 in method precision and 2.56 during the variation of analyst, system and column on a different day. The overall %RSD of 2.73 showed the ruggedness of the method. The GTI is linear from 0.039 to 2.9ppm (LOQ to 150%) and R= 0.9998 showed good linearity. The % recovery obtained for genotoxic impurity was in the range 103.0-109.6% with %RSD 1.51-3.19 from 0.42 to 2.5ppm. The %RSD in precision and accuracy at LOQ is 1.63 and 3.19 indicating the sensitivity of the method. The newly proposed and developed high-sensitive method can be used to detect GTI in Raltegravir API and formulation.

Determination of Three Alkyl Camphorsulfonates as Potential Genotoxic Impurities Using GC-FID and GC-MS by Analytical QbD

Camphorsulfonic acid salts are commonly used in the manufacturing production of active pharmaceutical ingredients (APIs) and have the potential to form alkyl camphorsulfonates, which can be considered as potential genotoxic impurities (PGIs). Alkyl camphorsulfonates should be controlled using the Threshold of Toxicological Concern (TTC) when detected in APIs due to their genotoxicity. An in silico study utilizing the ICH M7 guideline was performed in order to classify the alkyl camphorsulfonates that can be produced from the reaction of camphorsulfonic acid salts with methanol, ethanol, and isopropyl alcohol, which are commonly used solvents in API manufacturing processes. Two sensitive, reproducible, and accurate analytical methods using GC-FID and GC-MS were developed using the analytical Quality By Design (QbD) approaches for the quantitation of three alkyl camphorsulfonates in APIs satisfying the control limit of PGIs according to the TTC. The detection limits of the GC-FID method were found to be between 1.5 to 1.9 ppm, and the detection limits of the GC-MS method were found to be between 0.055 to 0.102 ppm. The method was validated in terms of accuracy, linearity, precision, detection limit, quantitation limit, specificity and robustness.

Functional group-specific multilateral derivatization cum extraction method for simultaneous quantification of genotoxic impurities in carvedilol phosphate drug using GC-MS and their toxicity assessments

A simple and facile functional group-specific multilateral derivatization cum extraction method coupled with GC-MS based analytical methodology has been developed for the rapid identification and determination of five potential genotoxic impurities (GTIs), including epichlorohydrin, hydrazine, phenylhydrazine, 3-chloro-1,2-propanediol and 1-(2-chloroethoxy)− 2-methoxybenzene in the carvedilol phosphate (CRV-P) drug active pharmaceutical ingredient (API). A generic synthetic route has been explored to apply the current investigation to the majority of the market available synthetic routes for the carvedilol process. Five significant GTIs were identified, and their toxicity was examined using in-silico model. The pharmacokinetic and pharmacodynamic properties of the impurities were compared with the drug molecule to evince the associated risk of impurities during therapeutic action. Furthermore, a quantitative comparison has been made for each impurity with the drug molecule for their ADMET properties, and the potential nature of the impurities has been thoroughly assessed. The developed method encompasses simple derivatization cum extraction-oriented GC-MS method for the reported GTIs, which was also validated as per current ICH guidelines. The obtained LOD and LOQ for the method were between 0.06 ~ 0.61 µg/g and 0.17 ~ 1.8 µg/g, respectively, and the r2 values (0.994 ~ 0.997) show that the method is very sensitive and linear over a wide range (LOQ to 120 % of the target concentration). The percentage recoveries and relative standard deviation obtained were between 85.3 and 109.5 and 0.1–4.7, respectively, showing fit for purpose. Moreover, method precision, intermediate precision, and robustness of the method have also been successfully demonstrated. Thus, this method could be directly engaged as the quality forecasting tool for the marketed drug samples aimed at the estimation of the reported GTIs at trace level.

Improved and ‘Nitrosamines Free’ Process for the Preparation of an α4β2 Neuronal Nicotinic Acetylcholine Receptor Agonist-Varenicline Tartrate

An improvised and efficient approach for synthesis of α4β2 nicotinic acetylcholine receptor subtype agonist, Varenicline tartrate (1) free from the ‘N-nitrosamines’ has been described. The approach involves an improved process for a key intermediate (7,8-dinitro-4,5-dihydro-1H-1,5-methanobenzo[d] azepin-3(2H)-yl)-2,2,2-trifluoro ethanone (7) free from potential genotoxic impurities. Compound 7 is converted into Varenicline base 10 in a single pot process with improved overall yield and quality. Further, Varenicline base 10 is converted into Varenicline tartrate (1) by acid addition salt which provides in quantitative yield. Our improved process consists of technical innovations/improvements which eliminate the probability for the formation of critical impurities such as dinitro nitroso impurity 2, diamino nitroso impurity 3 and varenicline nitroso impurity 4 and other genotoxic impurities such as mono nitro impurity 11 and meta dinitro impurity 12 in the final drug substance and provides ‘Nitrosamines free’ varenicline tartrate (1) with good quality and yield.

A Multi-Analyte LC-MS/MS Method for Determination and Quantification of Six Nitrosamine Impurities in Sartans like Azilsartan, Valsartan, Telmisartan, Olmesartan, Losartan and Irbesartan.

A sensitive and robust method for determination and quantification of potential genotoxic impurities in sartans has been developed. These impurities need to be controlled at trace levels during quantification in drug substances and drug products for safe consumption. Recent regulatory requirements also suggested the need to have highly sensitive analytical method for trace level quantification of nitrosamine impurities. In this paper, we have described a simple, rapid and sensitive liquid chromatography-mass spectrometry method for six potential genotoxic nitrosamine impurities: N-Nitroso dimethyl amine (NDMA), N-Nitroso diethyl amine (NDEA), N-Nitroso Ethyl Iso propylamine (NIPEA), N-Nitroso-Nmethyl-4-aminobutyric acid (NMBA) N-Nitroso diisopropylamino (NDIPA) and N-Nitroso dibutyl amine (NDBA) in Azilsartan (AZL), Valsartan (VAL), Telmisartan (TEL), Olmesartan (OLM), Losartan (LOS) and Irbesartan (IRB) with a limit of quantification of less than 0.003ppm. Chromatographic separation is achieved using Poroshell HPH- C18, 150 × 4.6mm, 2.7μm column with 0.1% formic acid in water as mobile phase A and 0.1% formic acid in methanol as mobile phase B at a flow rate of 0.5mL/min using gradient mode of elution at a total run time of 20min. Six nitrosamine impurities are ionized and quantified in positive mode of atmospheric pressure chemical ionization using multiple reaction monitoring. As per ICH guidelines, method validation is performed and evaluated the limit of quantification and detection and found to give good S/N ratios with good linearity range of 0.003-0.045ppm with regression coefficient > 0.999 for all the six nitrosamine impurities. Method recoveries are also established using three-step sample preparation and are found to be satisfactory within 80-120%. The single method can be used routinely applied for the detection of nitrosamines in AZL, VAL, TEL, OLM, LOS and IRB.

Analytical Method Development for 19 Alkyl Halides as Potential Genotoxic Impurities by Analytical Quality by Design

Major issues in the pharmaceutical industry involve efficient risk management and control strategies of potential genotoxic impurities (PGIs). As a result, the development of an appropriate method to control these impurities is required. An optimally sensitive and simultaneous analytical method using gas chromatography with a mass spectrometry detector (GC–MS) was developed for 19 alkyl halides determined to be PGIs. These 19 alkyl halides were selected from 144 alkyl halides through an in silico study utilizing quantitative structure–activity relationship (Q-SAR) approaches via expert knowledge rule-based software and statistical-based software. The analytical quality by design (QbD) approach was adopted for the development of a sensitive and robust analytical method for PGIs. A limited number of literature studies have reviewed the analytical QbD approach in the PGI method development using GC–MS as the analytical instrument. A GC equipped with a single quadrupole mass spectrometry detector (MSD) and VF-624 ms capillary column was used. The developed method was validated in terms of specificity, the limit of detection, quantitation, linearity, accuracy, and precision, according to the ICH Q2 guideline.

A sensitive LC-MS/MS method for the determination of potential genotoxic impurities in Cinnarizine

A highly sensitive LC-MS/MS method for the trace level determination of genotoxic impurities, Cinnamyl chloride and Benzhydryl chloride, in Cinnarizine drug substance was developed and validated. Chromatographic separation was successfully achieved on Atlantis d C18 column with dimensions 150× 4.6mm and particle size: 5μm. 0.1% Trifluoroacetic acid in water and 100% acetonitrile was used as mobile phases with gradient mode of elution at 1.0mL/min flow rate. Mass spectroscopic detection was carried out with selective ion monitoring (SIM) technique in positive mode at m/z 117 and 167 for Cinnamyl chloride and Benzhydryl chloride respectively. Developed method was proven to be selective, sensitive, and precise for the quantification of potential genotoxic impurities in Cinnarizine by validating as per the regulatory guidelines. The LOD and LOQ values observed for Cinnamyl chloride were 0.49 and 1.47ppm and for Benzhydryl chloride 0.55 and 1.67ppm respectively. Precision of the method at LOQ level was shown with good % RSD of 4.21. Method was proven linear from LOQ to 150% level with a correlation of 0.996 and accurate with a range of recovery from 86.4 to 100.8%. This highly sensitive method can be used to control both the genotoxic impurities in Cinnarizine drug substance by LC-MS/MS.

Quantitative Determination of Four Potential Genotoxic Impurities in the Active Pharmaceutical Ingredients in TSD-1 Using UPLC-MS/MS.

A novel method of ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed for the identification and quantification of four potential genotoxic impurities (PGIs) in the active pharmaceutical ingredients of TSD-1, a novel P2Y12 receptor antagonist. Four PGIs were named, 4-nitrobenzenesulfonic acid, methyl 4-nitrobenzenesulfonate, ethyl 4-nitrobenzenesulfonate, and isopropyl 4-nitrobenzenesulfonate. Following the International Conference of Harmonization (ICH) guidelines, this methodology is capable of quantifying four PGIs at 15.0 ppm in samples of 0.5 mg/mL concentration. This validated approach presented very low limits (0.1512–0.3897 ng/mL), excellent linearity (coefficients > 0.9900), and a satisfactory recovery range (94.9–115.5%). The method was sufficient in terms of sensitivity, linearity, precision, accuracy, selectivity, and robustness and, thus, has high practicality in the pharmaceutical quality control of TSD-1.

Simultaneous and trace level quantification of two potential genotoxic impurities in valsartan drug substance using UPLC-MS/MS

A sensitive and selective Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed for the identification and quantification of two potential genotoxic impurities (PGIs) - viz. methyl N–((2′–(1H–tetrazol-5-yl)-[1,1′-biphenyl]−4-yl)methyl)-N-nitroso-L-valinate (PGI-1) and N-nitroso Valsartan (PGI-2) - in the angiotensin II receptor blocker valsartan. Among these impurities, PGI-1 is a distinctive compound which has never been reported. For this, chromatographic separation was performed using a Waters XBridge BEH C18 column (150 mm × 4.6 mm, 2.5 µm), with ammonium acetate aqueous solution (0.01 mol/L) as mobile phase A and acetonitrile as mobile phase B, in a gradient elution mode at a 0.5 mL/min flow rate. Mass spectrometric conditions were optimized using electrospray ionization (ESI) in positive mode. Following the International Conference of Harmonization (ICH) guidelines, this methodology is capable of quantifying 2 PGIs at 0.016 ppm in samples at 50 mg/mL concentration. This validated approach presented good linearity over the concentration range of 0.016–0.06 ppm for 2 PGIs. The correlation coefficient of each impurity was observed greater than 0.999. The accuracy of this method was in the range of 83–113% for the aforementioned PGIs. In addition, expert knowledge rules (Derek-based) and statistical (Q) SAR evaluation system (Sarah-based) were used to evaluate and classify the genotoxicity of both valsartan-related PGIs as well as to define their standard limits. The predicted results were positive and classified into the third category, and the total nitrosamine limit was set to 0.03 ppm. As such, this approach represents a good quality control system for the simultaneous and precise quantitation of PGIs in valsartan.

High-performance thin-layer chromatography-based method development for the analysis of 4-methoxy-2-nitroaniline as potential genotoxic impurity

High-performance thin-layer chromatography-based method development for the analysis of 4-methoxy-2-nitroaniline as potential genotoxic impurity

Synthesis, characterization and control of eight process-related and two genotoxic fingolimod impurities by a validated RP-UPLC method.

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.

Toxicity estimation using QSAR methodology and analytical approach for the accurate determination of two potential genotoxic impurities in Abacavir Sulfate antiviral drug at TTC level by liquid chromatography-mass spectrometry (LC-MS/MS)

Toxicity estimation using QSAR methodology and analytical approach for the accurate determination of two potential genotoxic impurities in Abacavir Sulfate antiviral drug at TTC level by liquid chromatography-mass spectrometry (LC-MS/MS)

Detection and Quantification of Two Potential Genotoxic Impurities in Telbivudine Active Pharmaceutical Ingredient by Targeted Ultra Performance LC-MS/MS Analysis

A new selective, rapid and highly sensitive method using reversed-phase ultra-performance liquid chromatography (RP-UPLC) coupled with mass analyzer was developed for the detection and quantification of two genotoxic impurities viz. 4-dimethyl aminopyridine (GTI-1) and 2-bromo-3,5-diacetyl thymidine (GTI-2) in Telbivudine drug substance. The separation of two genotoxic impurities was accomplished using Kromasil C8 column (100 mm × 4.6 mm, 3.5 μm) maintained at 45 ºC using formic acid (0.1%) as buffer solution and gradient grade acetonitrile with 0.5 mL/min flow rate. Both GTIs were quantified using positive electrospray ionization (ESI) mode on MS/MS Analyser. ICH guidelines were followed for validation of genotoxic impurities. % RSD for system precision was found to be 2.71 and 2.94 and the overall %RSD for ruggedness was 3.11 and 2.89, respectively for GTI-1 and GTI-2. Two GTIs and Telbivudine drug were well resolved at retention times: 1.76 min for GTI-1, 3.81 min for Telbivudine and 6.62 min for GTI-2. The linearity relationship holds good in the range of concentrations 0.5 ppm to 7.5 ppm for both the impurities with a correlation coefficient of 0.999. Accuracy ranged from 95.1% to 105.1%. The GTI-1 and GTI-2 were shown, respectively detection limits: 0.07 ppm and 0.09 ppm and quantification limits: 0.22 ppm and 0.27 ppm and the solutions were stable upto 24 h at room temperature. The proposed and validated method is sensitive, reproducible, specific and linear and can be successfully applied for the quantification of both genotoxic impurities in Telbivudine active pharmaceutical ingredient and formulation.

Characterization of Loratadine API and Simultaneous Quantification of Seven Potential Genotoxic Nitrosamine Impurities in Single Method by LC-MS/MS in Loratadine API and its Dosage Forms

Active pharmaceutical ingredients (APIs) of loratadine were characterized using spectroscopic methods such as infrared spectroscopy, mass spectroscopy, differential scanning calorimetry (DSC), 1H NMR, 2D nuclear magnetic resonance (2D-NMR) and 13C NMR. Nitrosamine impurities, which are considered under concern cohort according to the S2 FDA and ICH M7 guidelines, are highly genotoxic and their trace-level quantity must be controlled in drugs for safe human consumption. In this study, an ultra-sensitive, rapid and simple LC-MS/MS technique is developed to detect seven nitrosamine impurities (N-nitroso dimethylamine (NDMA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), N-nitroso diethylamine (NDEA), N-nitroso ethyl isopropylamine (NEIPA), N-nitroso diisopropylamino (NDIPA), N-nitroso methyl phenylamine (NMPA) and N-nitroso dibutylamine (NDBA) in loratadine drug) with potential genotoxicity. Chromatographic separation was attained by employing the Zorbax SB C18 of 150 × 3 mm and a column of 3.5 μ with 0.1% formic acid in water and methanol as mobile phases A and B, respectively. At the total run time of 20 min, the flow rate was 0.3 mL/min in the gradient mode of elution. Through multiple reaction monitoring (MRM), all the seven nitrosamine impurities were successfully ionized and then quantified in the positive mode of atmospheric pressure chemical ionization (APCI). The method was validated according to the ICH guidelines by estimating quantification and detection limits. For all the seven nitrosamine impurities, the method provided excellent S/N ratios with a high linearity range of 0.8-5.30 ppm for loratadine sample concentrations with a regression coefficient of >0.99. The recovery for the method was established with a protocol of three-step sample preparation and was satisfactory within 15-115%. The proposed method can be employed for the routine detection of nitrosamines in loratadine APIs and its doses.

Determination of Potential Genotoxic Impurities (Orthophenylene Diamine Dihydrochloride), 4'-Bromomethyl-2-Cyanobiphenyl, 4'(Dibromomethyl)[1,1'-Biphenyl]-2-Carbonitrile in Telmisartan by ESI-MS/MS.

A sensitive, simple, rapid and robust LC-MS/MS method was developed for the determination of potential genotoxic impurities OPDA HCl (orthophenylene diamine dihydrochloride), bromo OTBN (4'-bromomethyl-2-cyanobiphenyl), dibromo (4'(dibromomethyl)[1,1'-biphenyl]-2-carbonitrile) in Telmisartan by using ESI-MS/MS Technic; the study was performed with gradient elution (time/% mobile phase B: 0/10, 3/10, 30/80, 35/80, 36/10, 40/10). The mobile phase consisted of a mixture of formic acid, methanol and acetonitrile. The buffer was degassed before running at a flow rate of 1.0mL/min. The column temperature was at 40°C. The 20μL volume of sample was injected per run and peaks were detected using DAD detector. The LC-ESI/MS/MS studies were carried out on Ultivo Triple Quadrupole LC/MS/MS (Agilent, USA) G6470A mass spectrometer, ion source voltage 3500V, de clustering potential 40V, entrance potential 10V, with the nebulizer gas as nitrogen at 45psi. The LC part consisted of Agilent 1260 series HPLC system with binary gradient pump with a degasser and an auto sampler. Inert sustain AQ-C18, 250 × 4.6mm, 5-μm particle size was used for chromatographic separation. Developed method is validated as per ICH guidelines and found to be linear, accurate, specific, selective, precise and robust. Test solution was found to be stable up to 48h. This method can be successfully applied for the determination of genotoxic impurities in Telmisarta for routine analysis and stability assessment.

Sensitive and Selective Analytical Method for the Quantification of Two Potential Genotoxic Impurities in Azilsartan Drug Substance by using LC-MS/MS with Multiple Reaction Monitoring (MRM mode)

The main aim of the present study to Synthesize,method development and method validation for quantification of two potential genotoxic impurities i.e., Methyl(Z)-2-ethoxy-1-((2'-(N'-hydroxycarbamimidoyl)-[1,1'-biphenyl]-4-yl) methyl)-1H-benzo[d]imidazole-7-carboxylate(Impurity-A) and 2-ethoxy-1-((2'-(N'-hydroxycarbamimidoyl[)-[1,1'-biphenyl]-4-yl)methyl)-1H-benzo[d]imidazole-7-carboxylicacid(Impurity-B) by using LC-MS/MS MRM mode at trace level determination inAzilsartan drug substance. The new LC-MS/MS MRM mode method was developed by using Inertsil ODS-3V 150x4.6mm,5µm column as stationary phase. The mobile phase used is the composition of 10Mm Ammonium formate pH3.00buffer:Acetonitrile(9:1)%v/v as mobilephase-A and 10Mm Ammonium formate pH3.00buffer:Acetonitrile(2:8)%v/v as mobile phase-B, isocratic elution of mobile phase-A and Mobile phase-B (60:40)v/v at flow rate of 0.8mL/min.The concentration limits of the both genotoxic impurities were calculated a limit of 37.5ppm based on the concept of TTC (threshold of toxicological concern) and MDD (maximum daily dosage which is 40mg/day for Azilsartan drug substance.The limit of detection(LOD) was found to be 1.4ppm for both Impurity-A and Impurity-B.The limit of quantification(LOQ) for Impurity-A was 4.7ppm and Impurity-B was 4.5ppm respectively The method was found to be linear from 4.7ppm to 78.4ppm for Impurity-A (correlation coefficient:1.000) and 4.5ppm to 75.7ppm for Impurity-B (correlation coefficient:0.999). The method was precise and found percentage of relative standard deviation for six replicate sample preparations of Impurity-A and Impurity-B was below 5.0%. The method accuracy was confirmed based on the recovery studies. Based on the method validation study method was sensitive and selective for quantification both genotoxic impurities.

Development of GC Method for Analyzing Potential Genotoxic Impurities at Low-level Determination in Atorvastatin Calcium

Atorvastatin Calcium (ATC) is a specific HMG-CoA reductase inhibitor utilized in the avoidance and treatment of cardiovascular infection in those at high danger and treat strange lipid levels. For the quantitative analysis of four PGIs in the medicine ingredient ATC, an ICH-approved Gas chromatography (GC) approach has been developed. Within the examined fixation range, the novel approach was explicit, exact, precise, and direct. Pollutant D, with 0.6 ng/mL, had the lowest recognition level among the four PGIs, which were all below 10 ng/mL. Every contaminant from ATC has been effectively chromatographically separated into its component parts. At a low level, ATC's technique was found to be highly effective in terms of pollution reduction. Proposed techniques are relied upon to measure the strength of ATC creation and to examine PGIs as a reference for ATC assessments. This method can be used to check for pollution levels in the manufacturing process of pharmaceuticals. The API's PGI levels will be kept low thanks to the GC's oversight. As a result, this study's findings will help ensure the safe use of APIs during clinical therapy.

Determination of potentially genotoxic impurities in crotamiton active pharmaceutical ingredient by gas chromatography

In the recent years control of potentially genotoxic impurities have an increasing importance in the analysis of active pharmaceutical ingredients. Guidelines of different regulatory bodies specify very low limits for these impurities, in many cases the analytical development is challenging to comply with the requirements. In this paper potential genotoxic impurities of Crotamiton drug substance are investigated, a simple and robust gas chromatographic method is developed for the determination of genotoxic impurities in Crotamiton drug substance. One of the main benefits of this method is that it can quantify all potential genotoxic impurities that can be present in the entire synthesis pathway of Crotamiton drug substance. Crotamiton is synthesized from toluidine derivatives, in which the aromatic amine group represents genotoxic alert. In the method development toluidine isomers (o-, m-, p-toluidine), N-ethyl-toluidine isomers (o-, m-, N-ethyl-p-toluidine) and N-methyl-toluidine isomers (o-, m-, N-methyl-p-toluidine) were separated, and the developed gas chromatographic method is validated in accordance with the relevant guidelines at a specification limit of NMT 40 ppm.

Liquid chromatography-mass spectrometric methods for trace quantification of potential genotoxic impurities in ivacaftor and lumacaftor

The objective of the current study was to develop and validate the sensitive LC-MS methods for trace analysis of genotoxic impurities in Ivacaftor and Lumacaftor. The first method is for the trace analysis of 2,4-di-tert-butyl-5-nitrophenol in ivacaftor and the second method is for the trace analysis of 1-(2,2-difluoro-1,3-benzodioxol-5yl)-cyclopropane carboxylic acid and 3-carboxyphenyl boronic acid in lumacaftor.High pure analytical grade solvents and reagents were used for this study. The chromatographic separation was performed on Luna C18 (250×4.6mm, 5.0μm) at a column temperature of 25°C using eluent consisting of acetonitrile and 0.1% v/v formic acid in water in a gradient elution mode. The eluent was run at a flow of 1.0mL/min and injection volume of 20μL.The linearity, precision and accuracy of the developed methods was validated over the concentration range of 0.35-15.0ppm for 2,4-di-tert-butyl-5-nitrophenol, 0.30-15.0ppm for 1-(2,2-difluoro-1,3-benzodioxol-5yl)-cyclopropane carboxylic acid and 0.23-15.0ppm for 3-carboxyphenyl boronic acid. In both methods, interference was not observed at the retention time of analyte peaks. All the analytes were found to be stable in solution for a period of 48h.The proposed methods are reliable, sensitive, precise, accurate, and robust for the trace level quantification of genotoxic impurities in Ivacaftor and Lumacaftor. These methods can be successfully implemented in the quality control lab for routine analysis.

HPLC-MS Analysis of Four Potential Genotoxic Impurities in Alogliptin Pharmaceutical Materials.

Pyridine, 3-aminopyridine, 4-dimethylaminopyridine, and N, N-dimethylamine are reactive bases that may be used in the preparation of the pharmaceutical ingredient alogliptin (ALO). They are considered as potentially genotoxic impurities (PGIs) since they contain electrophilic functional groups. Therefore, they should be monitored at the allowed limits in ALO. The aim of this study was to develop a novel liquid chromatography-mass spectrometry (LC-MS) method to estimate quantities of pyridine, 3-aminopyridine, 4-dimethylaminopyridine, and N, N-dimethylaniline impurities in ALO drug material. The separation was performed on Kromasil CN (250 mm × 3.9 mm, 3.5 µm) column in reversed phase mode. The mobile phase was a mixture of water-methanol (55:45, v/v) containing 2.5 mM ammonium acetate and 0.1% formic acid. Impurities were detected by MS in selected ion monitoring mode at m/z = 80, 95, 122, and 123 for pyridine, 3-aminopyridine, N, N-dimethylaniline, and 4-dimethylaminopyridine, respectively. The flow rate of the method was 0.5 mL/min. The sensitivity of the method was excellent at levels much less than the allowed limits. The method had excellent linearity in the concentration ranges of Quantitation Limit (QL)-150% of allowed limits and coefficients of determination were above 0.9990. The recovery ratios were in the range of 93.56-110.28%. Results showed good linearity, precision, accuracy, sensitivity, selectivity, robustness, and solution stability. The studied method was applied to test two samples of raw materials and one sample of tablets. The method discussed here could be very useful for controlling potentially genotoxic impurities (PGIs) levels in ALO during its synthesis, and for QC testing of ALO raw materials before using them in the preparation of pharmaceutical products.