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

Ali S.M., Moorthy M.K., Devanna N.

AbstractIn the present study, the main objective is to develop an analytical method for ultra‐trace level measurement of 2,6‐diamino‐5‐nitropyrimidin‐4(3H)‐one (DMNP) in valganciclovir hydrochloride (VAL) using liquid chromatography‐quadrupole time‐of‐flight‐tandem mass spectroscopy (LC–QTOF–MS/MS). In the early stages of guanine synthesis, DMNP is formed, and guanine is known to be the key starting material for the synthesis of VAL. Taking into consideration DMNP potential genotoxicity, this analytical method has been developed. This method is time saving and suitable for confirming the masses of parent and fragment ions by MS and MS/MS further fragmentation. An isocratic program and Acquity UPLC HSS cyano column (100 × 2.1 mm × 1.8 μm) were used to achieve optimal separation between VAL and the DMNP impurity. A 0.1% ammonia solution in Milli‐Q water was used as mobile phase A, and methanol was used as mobile phase B in the ratio 90:10 v/v in isocratic mode. In accordance with the International Conference on Harmonization's requirements, the developed method was validated. The detection and quantification levels were found to be 0.028 and 0.083 ppm respectively. The DMNP impurity is linear from 0.083 to 1.245 ppm levels with correlation coefficient (R2) of 0.9960. The recoveries were found to be 97.0–107.9%.

Shaik M.A., Manchuri K.M., Nayakanti D.

Mullangi S., Ravindhranath K., Yarala M.R., Panchakarla R.K.

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.

Moorthy M.K., Ali S.M., Reddy G.V.

The foremost objective of the present study was to develop and validate a new LC–QTOF–MS/MS method for the identification and quantitative determination of 4,6-dichloro-5-nitro-2-(propylthio)pyrimidine (DPP) genotoxic impurity through the derivatization process in ticagrelor active pharmaceutical ingredient (API). Owing to the low response of DPP at the specification level, DPP was converted to 4,6-dibenzylamino-5-nitro-2-(propylthio)pyrimidine (DPP derivative) by addition of benzyl amine, then analyzed using mass spectrometry with a time-of-flight analyzer, and good separation was accomplished under the experimental conditions described. The effective separation of DPP derivative was achieved using an Acquity UPLC BEH C18 reverse-phase column (100 × 4.6 mm × 1.7 μm) with an isocratic program with mobile phase A as 0.1% formic acid in milli Q water and mobile phase B as acetonitrile in the ratio of 20:80 v/v. The flow rate was maintained as 0.4 ml/min, the injection volume was 2 μl, the autosampler temperature was 5°C, the column oven temperature was ambient and the run time was 6.0 min. The diluent used was 0.2% benzyl amine in water and acetonitrile in the ratio of 30:70 v/v. The retention time of the DPP derivative was 2.87 min. The limit of detection and limit of quantification were 0.03 and 0.08 ppm, respectively. The DPP derivative was linear from 1.68 to 12.78 ppm with R2 of 0.9958. Thus, the developed method is valid for the identification and quantitative determination of DPP derivative in ticagrelor API.

Manchuri K.M., Shaik M.A., Gopireddy V.S.

A selective and sensitive novel reverse-phase method was developed and validated successfully using waters acquity ultra-performance liquid chromatography H-class system coupled with waters Micromass Quattro Premier XE (UHPLC–MS/MS) for the identification and quantification of bis (2-chloroethyl) amine genotoxic impurity in aripiprazole drug substance. The developed method employs a positive-ion electrospray ionization (ESI) with multiple reaction monitoring (MRM) detection mode using ACE 3 C18 (100 mm × 4.6 mm × 3.0 µm) column. An isocratic program was developed for the rapid analysis using 0.2% formic acid (mobile phase-A) in milli Q water and methanol (mobile phase-B) in the ratio of 45:55 v/v. The elution of bis (2-chloroethyl) amine monitored using triple quadrupole mass spectrophotometer with injection volume 2 μL, column oven temperature 40 °C, auto-sampler temperature 15 °C, and flow rate 0.5 mL/min. The retention time of bis (2-chloroethyl) amine was observed at 2.00 min. The limit of detection and the limit of quantification were observed at concentration levels of 0.070 ppm and 0.206 ppm, respectively. The correlation coefficient (R2) obtained was 0.9892 and the percentage recoveries of the method was between the ranges of 92.0–111.0%. The method was successfully developed and validated according to the current requirements of International Council for Harmonization (ICH Q2 (R1) and ICH M7 (R1)) for genotoxic impurities. The developed method was sensitive, selective, rapid, accurate, and precise, and, therefore, can be used for the identification and quantification of bis (2-chloroethyl) amine genotoxic impurity in aripiprazole drug substance.

Moorthy M.K., Ali S.M., Reddy G.V.

A new reverse phase and shorter run time (3.0 min) liquid chromatography–quadrupole time of flight-tandem mass spectrometry method was developed and validated for identification and ultra-trace level quantification (0.83 ppm) of genotoxic impurity 1,3-diacetoxy-2-(acetoxymethoxy) propane in valganciclovir hydrochloride active pharmaceutical ingredient. The method is cost effective, time saving and proficient to confirm the parent and fragment ion masses through mass spectrometry and tandem mass spectrometry further fragmentation. An isocratic program and acquity bridged ethylene hybrid C18 reverse phase column (100 mm × 4.6 mm × 1.7 μm) was used to accomplish optimum separation between valganciclovir and 1,3-diacetoxy-2-(acetoxymethoxy) propane impurity. Mobile phase-A used was 0.1% formic acid in milli Q water and mobile phase-B used was acetonitrile in the ration of 50:50 v/v. Diluent used was water and methanol in the ratio of 30:70 v/v. Chromatographic conditions are selected as injection volume: 3 μL, flow rate: 0.2 mL/min, oven temperature: ambient, auto sampler: 5°C and run time: 3.0 min. The retention time of 1,3-diacetoxy-2-(acetoxymethoxy) propane impurity was found at 1.830 min. The detection and quantification levels found at 0.027 and 0.083 ppm. The 1,3-diacetoxy-2-(acetoxymethoxy) propane impurity is linear from 0.082 to 1.236 ppm levels with regression coefficient 0.9972. The recoveries were from 93.3 to 110.0%.

Kelemen H., Hancu G., Papp L.A.

Ticagrelor is an orally administered platelet aggregation inhibitor with a cyclopentyl-triazolopyrimidine structure; it is a selective reversible P2Y12 receptor antagonist, which prevents P2Y12-mediated and ADP-mediated platelet activation and aggregation. It is used to reduce the rate of cardiovascular death, myocardial infarction and stroke in patients with acute coronary syndrome or history of myocardial infarction. Several analytical methods have been published for the determination of ticagrelor in pharmaceuticals and biological materials by spectrophotometry, high-performance liquid chromatography with ultraviolet detection and liquid chromatography coupled with tandem mass spectrometry. The purpose of the current review is to provide a systematic survey of the analytical techniques used for the determination of ticagrelor since its introduction in therapy until today.

Wingert N.R., Ellwanger J.B., Bueno L.M., Gobetti C., Garcia C.V., Steppe M., Schapoval E.E.

Simultaneous analysis of drug compounds and their impurities of degradation and synthesis became constant in the modern pharmaceutical analysis. Likewise, analytical techniques must improve sensitivity and selectivity for the monitoring of pharmaceutical products, allowing a full assessment of impurities in drug products and, therefore, ensure safety and efficacy of pharmacological treatments. The application of Quality by Design (QbD) principles has proved to be feasible on the elaboration of analytical methods, allowing the comprehensive evaluation and measurement of different analytical parameters and their effects on critical properties of the methodology in development. QbD approach was applied to the development of a fast and selective HPLC method for the analysis of the antiplatelet aggregation drug ticagrelor and its degradation products in presence of three impurities of synthesis. Fractional factorial resolution V was the screening experimental design applied to five method parameters. Response surface methodology was carried by central composite star face design on the two critical method parameters selected. Analytical design space, established after the application of Monte-Carlo simulations, verified whether predicted results were in accordance with critical quality attributes. The developed and validated HPLC method with DAD detection at 225 nm was able to resolve eight related compounds in less than three minutes.

Kakde R.B., Satone D.D., Dongare G., Chilbule R., Malkhede Y.

Tabassum K., Sarvesh R.

Objective: The present study was conducted to develop a simple and precise analytical method for the estimation of ticagrelor in tablet formulation.Methods: Reverse Phase HPLC was used for method development and validation studies of ticagrelor. The optimum chromatographic conditions comprised of C18 column (Kromasil, 250×4.6 mm, 5 µ) as the stationary phase and aqueous buffer (containing 0.5 ml formic acid and triethylamine each in water) and acetonitrile in the ratio of 50:50 v/v as the mobile phase. The flow rate was 1.3 ml/min with detection at 256 nm and a run time of 6 min. Forced degradation studies were conducted and the isocratic mode was modified to a gradient mode to make the method stability indicating in nature.Results: The retention time of ticagrelor was 3.372 min. The linearity studies indicated that the range of the developed method was 20-90 ppm with a correlation coefficient of 0.9956. The method was specific with a percent mean recovery was found to be 99.93%.. The % RSD in the precision studies was 0.069. The validated method was applied to conduct the assay of ticagrelor in tablets and the with a percent mean recovery of 99.82%. The modified method was capable of resolving 13 related substances from the ticagrelor peak in the forced degradation studies.Conclusion: The developed and validated RP-HPLC isocratic method was simple, accurate and precise as per the ICH guidelines. It was suitable for the analysis of ticagrelor in bulk and tablet formulation. The modified gradient method can be used to resolve the in-process impurities and related substances and can be directly applied to liquid chromatography hyphenated with mass spectroscopy (LC/MS) studies with minor modifications for identification of related substances.

Tan Q., Jiang X., Huang S., Zhang T., Chen L., Xie S., Mo E., Xu J., Cai S.

Objective In this study, a systematic evaluation was conducted to estimate the efficacy and safety of ticagrelor for treating acute coronary syndrome (ACS) in general ACS patients and a diabetes mellitus (DM) group. Methods A search of PubMed, Cochrane Central Register of Controlled Trials, Web of Science, CNKI databases was conducted to analyze relevant randomized controlled trails (RCTs) of ticagrelor treating ACS during 2007 to 2015. Article screening, quality accessing and data extracting was independently undertaken by two reviewers. A meta-analysis was performed to clarify the efficacy and safety of ticagrelor in general ACS patients, and a meta-regression analysis was taken to demonstrate the efficacy and safety of ticagrelor in DM patients compared with general ACS patients. Result Twenty-two studies with 35004 participants were included. The meta-analysis result implicated that ticagrelor could: 1) reduce the incidence of the composite endpoint [OR = 0.83, 95%CI (0.77, 0.90), P

Bueno L.M., Manoel J.W., Giordani C.F., Mendez A.S., Volpato N.M., Schapoval E.E., Steppe M., Garcia C.V.

A simple, fast and sensitive analytical method by high-performance liquid chromatography (HPLC) was developed and validated for the simultaneous determination of ticagrelor and two synthesis impurities. The HPLC method was established using an Agilent 1200 Series equipment coupled to photodiode array detector (PDA) at 270nm with a Zorbax Plus C8 column (150×4.6mm, 5.0μm), injection volume of 20μL, and a constant temperature of 25°C. The mobile phase consisted of acetonitrile: ammonium acetate 50mM (57:43, v/v) and pH adjusted to 8.2 with ammonium hydroxide 6M, at a flow rate of 0.7mL/min. No interference peaks from excipients and diluent system indicated the specificity of the method. The calibration curves showed determination coefficients (r2)>0.99, calculated by linear regression. The limit of quantitation (LOQ) for impurities 1 and 2 were 2.0 and 0.2μg/mL, respectively. Intra and interday relative standard deviations (RSDs) were

Li Z., Yang D., Geng P., Zhang J., Wei H., Hu B., Guo Q., Zhang X., Guo W., Zhao B., Yu B., Ma L., Liu H.

A series of [1,2,3]triazolo[4,5-d]pyrimidine derivatives bearing a hydrazone moiety were designed, synthesized and evaluated for their antiproliferative activity against several cancer cell lines of different origins by MTT assay. Most of the synthesized compounds demonstrated moderate to good activity against the cancer cell lines selected. Especially, compound 43 showed the most potent antiproliferative activity as well as good selectivity between cancer and normal cells (IC50 values of 0.85 μM against MGC-803 and 56.17 μM against GES-1). In addition, compound 43 evidently inhibited the colony formation of MGC-803 cells at 0.8 μM. Further mechanism studies revealed that compound 43 could induce apoptosis of MGC-803 cells probably through the mitochondrial pathway accompanied with decrease of the mitochondrial membrane potential (MMP), activations of caspase-9/3, up-regulation of the expression of Bax, Bak and PUMA, as well as down-regulation of that of Bcl-2 and Mcl-1.

Amberg A., Beilke L., Bercu J., Bower D., Brigo A., Cross K.P., Custer L., Dobo K., Dowdy E., Ford K.A., Glowienke S., Van Gompel J., Harvey J., Hasselgren C., Honma M., et. al.

The ICH M7 guideline describes a consistent approach to identify, categorize, and control DNA reactive, mutagenic, impurities in pharmaceutical products to limit the potential carcinogenic risk related to such impurities. This paper outlines a series of principles and procedures to consider when generating (Q)SAR assessments aligned with the ICH M7 guideline to be included in a regulatory submission. In the absence of adequate experimental data, the results from two complementary (Q)SAR methodologies may be combined to support an initial hazard classification. This may be followed by an assessment of additional information that serves as the basis for an expert review to support or refute the predictions. This paper elucidates scenarios where additional expert knowledge may be beneficial, what such an expert review may contain, and how the results and accompanying considerations may be documented. Furthermore, the use of these principles and procedures to yield a consistent and robust (Q)SAR-based argument to support impurity qualification for regulatory purposes is described in this manuscript.

Kumar N., Devineni S.R., Gajjala P.R., Gupta D.K., Bhat S., Kumar R., Dubey S.K., Kumar P.

Five process-related impurities were detected in the range of 0.08-0.22% in ticagrelor laboratory batches by HPLC and LC-MS methods. These impurities were named as TIC Imp-I, -II, -III, -IV and -V. Four of these impurities, TIC Imp-I to -IV were unknown and have not been reported previously. Based on LC-ESI/MS(n) study, the chemical structures of new impurities were presumed as (1S,2S,3S,5S)-3-(2-hydroxyethoxy)-5-(7-amino-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d] pyrimidin-3-yl)cyclopentane-1,2-diol (TIC Imp-I), (1S,2S,3S,5S)-3-(7-((1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino)-5-(propylsulfinyl)-3H-[1,2,3]triazolo [4,5-d]pyrimidin-3-yl)-5-(2-hydroxyethoxy)cyclopentane-1,2-diol (TIC Imp-II), (1S,2R,3S,4S)-4-(7-((1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)cyclopentane-1,2,3-triol (TIC Imp-III) and (3S,5S)-3-(7-((1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-5-(2-hydroxyethoxy)cyclopentane-1,2-diol (TIC Imp-IV). The unknown impurities were isolated from enriched crude sample by column chromatography and preparative HPLC. The complete spectral analysis, MS, 1D NMR ((1)H, (13)C and DEPT), 2D NMR (HSQC and HMBC) and IR confirmed the proposed chemical structures of impurities. Identification, isolation, structural characterization, prospects for the formation of impurities and their synthesis were first reported in this paper.

Patel H.B., Dave R.H., Vadariya S., Hirpara H., Patel T.

Manchuri K.M., Shaik M.A., Gopireddy V.S., Naziya Sultana, Gogineni S.