Characterization of forced degradation products of canagliflozine by liquid chromatography/quadrupole time-of-flight tandem mass spectrometry and in silico toxicity predictions.

RATIONALE: Forced degradation studies are useful for better understanding of the stability of active pharmaceutical ingredients and drugs and to generate information about drug degradation pathways and formation of degradation products (DPs). Identification of DPs plays a vital role in establishing the safety and therapeutic benefit of a drug. METHODS: Canagliflozin (CAN) was subjected to different stress conditions as per International Conference on Harmonization guidelines (Q1A R2). All the DPs and the drug were well separated on an Aquity CSH C18 (100 × 2.1 mm, 1.7 µm) column using acetonitrile-methanol (70:30, v/v) and formic acid in gradient mode. The same UPLC method was employed for LC/HRMS for the characterization of DPs. In addition, in silico toxicity was predicted for all the DPs by using TOPKAT and DEREK software tools. RESULTS: CAN was found to degrade under oxidative stress condition and formed DP1 and DP2. This is a typical case of degradation where co-solvents acetonitrile-water (50:50, v/v) and methanol-water (50:50, v/v) react with CAN under acid hydrolytic conditions leading to the formation of pseudo-DPs, DP3 and DP4, respectively. Among these, DP2 and DP3 showed ocular irritancy whereas DP1 showed skin sensitization. CONCLUSIONS: The drug was labile under oxidative stress condition. CAN reacted with co-solvent under acid hydrolytic conditions and gave pseudo-DPs. All the DPs were separated using UPLC and characterized by LC/QTOF/MS/MS. Toxicity of DPs was evaluated using TOPKAT and DEREK software tools.

Identification and characterization of vilazodone metabolites in rats and microsomes by ultrahigh-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry.

RATIONALE: Vilazodone is a selective serotonin reuptake inhibitor (SSRI) used for the treatment of major depressive disorder (MDD). An extensive literature search found few reports on the in vivo and in vitro metabolism of vilazodone. Therefore, we report a comprehensive in vivo and in vitro metabolic identification and structural characterization of vilazodone using ultrahigh-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF/MS/MS) and in silico toxicity study of the metabolites. METHODS: To identify in vivo metabolites of vilazodone, blood, urine and faeces samples were collected at different time intervals starting from 0 h to 48 h after oral administration of vilazodone to Sprague-Dawley rats. The in vitro metabolism study was conducted with human liver microsomes (HLM) and rat liver microsomes (RLM). The samples were prepared using an optimized sample preparation approach involving protein precipitation followed by solid-phase extraction. The metabolites have been identified and characterized by using LC/ESI-MS/MS. RESULTS: A total of 12 metabolites (M1-M12) were identified in in vivo and in vitro matrices and characterized by LC/ESI-MS/MS. The majority of the metabolites were observed in urine, while a few metabolites were present in faeces and plasma. Two metabolites were observed in the in vitro study. A semi-quantitative study based on percentage counts shows that metabolites M11, M6 and M8 were observed in higher amounts in urine, faeces and plasma, respectively. CONCLUSIONS: The structures of all the 12 metabolites were elucidated by using LC/ESI-MS/MS. The study suggests that vilazodone was metabolized via hydroxylation, dihydroxylation, glucuronidation, oxidative deamination, dealkylation, dehydrogenation and dioxidation. All the metabolites were screened for toxicity using an in silico tool.

In vivo metabolite identification of acotiamide in rats using ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry.

Acotiamide hydrochloride (ACT) is a drug used for the treatment of functional dyspepsia. Understanding which metabolites are likely to be formed in vivo is essential for interpreting pharmacology, pharmacokinetic and toxicology data. The metabolism of ACT has been investigated using a specific and sensitive liquid chromatography positive ion electrospray ionization high-resolution tandem mass spectrometry method. In vivo samples including rat plasma, urine and feces were collected separately after dosing healthy Sprague-Dawley rats at a dose of 20 mg kg -1 ACT at different time points up to 24 h. The metabolites were enriched by optimized sample preparation involving protein precipitation using acetonitrile followed by solid-phase extraction. The mass defect filter technique was used for better detection of both predicted and unexpected drug metabolites with the majority of interference ions removed. The structural elucidation of the metabolites was performed by comparing their [M + H]+ ions and their product ions with those of the parent drug. As a result, a total of seven hitherto unknown metabolites were characterized from the biosamples. The only phase I metabolite detected was N-despropyl acotiamide, whereas six phase II glucuronide conjugate metabolites were identified.

Automated statistical experimental design approach for rapid separation of coenzyme Q10 and identification of its biotechnological process related impurities using UHPLC and UHPLC-APCI-MS.

A novel ultra high performance liquid chromatography method development strategy was ameliorated by applying quality by design approach. The developed systematic approach was divided into five steps (i) Analytical Target Profile, (ii) Critical Quality Attributes, (iii) Risk Assessments of Critical parameters using design of experiments (screening and optimization phases), (iv) Generation of design space, and (v) Process Capability Analysis (Cp) for robustness study using Monte Carlo simulation. The complete quality-by-design-based method development was made automated and expedited by employing sub-2 µm particles column with an ultra high performance liquid chromatography system. Successful chromatographic separation of the Coenzyme Q10 from its biotechnological process related impurities was achieved on a Waters Acquity phenyl hexyl (100 mm × 2.1 mm, 1.7 µm) column with gradient elution of 10 mM ammonium acetate buffer (pH 4.0) and a mixture of acetonitrile/2-propanol (1:1) as the mobile phase. Through this study, fast and organized method development workflow was developed and robustness of the method was also demonstrated. The method was validated for specificity, linearity, accuracy, precision, and robustness in compliance to the International Conference on Harmonization, Q2 (R1) guidelines. The impurities were identified by atmospheric pressure chemical ionization-mass spectrometry technique. Further, the in silico toxicity of impurities was analyzed using TOPKAT and DEREK software.

Quantitation of acotiamide in rat plasma by UHPLC-Q-TOF-MS: method development, validation and application to pharmacokinetics.

A novel, sensitive and selective ultra-high-performance liquid chromatography-electrospray ionization mass spectrometry method was developed and validated for the quantification of acotiamide (ACT), a first-in-class drug used in functional dyspepsia, in rat plasma. A simple protein precipitation method with acetonitrile as precipitating solvent was used to extract ACT from rat plasma. ACT and an internal standard (mirabegron, IS) were separated on an Agilent poroshell EC C18 column (50 × 3.0 mm, 2.7 µm) using methanol-10 mM ammonium acetate binary gradient mobile phase at a flow rate of 0.4 mL/min over 4 min run time. Detection was performed using target ions of [M + H](+) at m/z 451.2010 for ACT and m/z 397.1693 for IS in selective ion mode. The method was validated in the calibration range of 1.31-1000 ng/mL. All the validation parameters were well within the limits. The method demonstrated good performances in terms of intra- and inter-day precision (3.27-12.60% CV) and accuracy (87.96-104.94%). Thus the present ultra-high-pressure liquid chromatograhy-high-resolution mass spectrometry method for determination of ACT in rat plasma, is highly sensitive and rapid with a short run-time of 4 min, can be suitable for high sample throughput and for large batches of biological samples in pharmacokinetic studies. This method can be extended to measure plasma concentrations of ACT in humans to understand drug metabolism, drug interaction and adverse effects.

Selective separation and characterization of the stress degradation products of ondansetron hydrochloride by liquid chromatography with quadrupole time-of-flight mass spectrometry.

Ondansetron hydrochloride was subjected to forced degradation studies under various conditions of hydrolysis (acidic, basic, and neutral), oxidation, photolysis, and thermal as prescribed by International Conference on Harmonisation guideline Q1A (R2). A simple, selective, precise, and accurate high-performance liquid chromatography method was developed on a Waters Xterra C18 (150 × 4.6 mm id, 3.5 µm) column using 10 mM ammonium formate (pH 3.0)/methanol as a mobile phase in gradient elution mode at a flow rate of 0.6 mL/min. The method was extended to liquid chromatography quadrupole time-of-flight tandem mass spectrometry for identification and structural characterization of stress degradation products of ondansetron. The drug showed significant degradation in base hydrolytic and photolytic stress conditions in the liquid state, while it was found to be stable in neutral, acidic, thermal, and oxidative stress conditions. A total of five degradation products were characterized and most probable mechanisms for the formation of degradation products have been proposed on the basis of a comparison of the fragmentation of the [M + H](+) ions of the drug and its degradation products. Finally, the developed method was validated in terms of specificity, linearity, accuracy, precision, and robustness as per International Conference on Harmonisation guideline Q2 (R1).

Quality by design: a systematic and rapid liquid chromatography and mass spectrometry method for eprosartan mesylate and its related impurities using a superficially porous particle column.

The present work describes the systematic development of a robust, precise, and rapid reversed-phase liquid chromatography method for the simultaneous determination of eprosartan mesylate and its six impurities using quality-by-design principles. The method was developed in two phases, screening and optimization. During the screening phase, the most suitable stationary phase, organic modifier, and pH were identified. The optimization was performed for secondary influential parameters--column temperature, gradient time, and flow rate using eight experiments--to examine multifactorial effects of parameters on the critical resolution and generated design space representing the robust region. A verification experiment was performed within the working design space and the model was found to be accurate. This study also describes other operating features of the column packed with superficially porous particles that allow very fast separations at pressures available in most liquid chromatography instruments. Successful chromatographic separation was achieved in less than 7 min using a fused-core C18 (100 mm × 2.1 mm, 2.6 µm) column with linear gradient elution of 10 mM ammonium formate (pH 3.0) and acetonitrile as the mobile phase. The method was validated for specificity, linearity, accuracy, precision, and robustness in compliance with the International Conference on Harmonization Q2 (R1) guidelines. The impurities were identified by liquid chromatography with mass spectrometry.

Identification and characterization of fluvastatin metabolites in rats by UHPLC/Q-TOF/MS/MS and in silico toxicological screening of the metabolites.

The present study reports the in vivo and in vitro identification and characterization of metabolites of fluvastatin, the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitor, using liquid chromatography-mass spectrometry (LC-MS). In vitro studies were conducted by incubating the drug with human liver microsomes and rat liver microsomes. In vivo studies were carried out by administration of the drug in the form of suspension to the Sprague-Dawley rats followed by collection of urine, faeces and blood at different time points up to 24 h. Further, samples were prepared by optimized sample preparation method, which includes freeze liquid extraction, protein precipitation and solid phase extraction. The extracted and concentrated samples were analysed using ultrahigh-performance liquid chromatography-quadruple time-of-flight tandem mass spectrometry. A total of 15 metabolites were observed in urine, which includes hydroxyl, sulphated, desisopropyl, dehydrogenated, dehydroxylated and glucuronide metabolites. A few of the metabolites were also present in faeces and plasma samples. In in vitro studies, a few metabolites were observed that were also present in in vivo samples. All the metabolites were characterized using ultrahigh-performance liquid chromatography-quadruple time-of-flight tandem mass spectrometry in combination with accurate mass measurement. Finally, in silico toxicity studies indicated that some of the metabolites show or possess carcinogenicity and skin sensitization. Several metabolites that were identified in rats are proposed to have toxicological significance on the basis of in silico evaluation. However, these metabolites are of no human relevance. Copyright © 2017 John Wiley & Sons, Ltd.

Selective separation and characterisation of stress degradation products and process impurities of prucalopride succinate by LC-QTOF-MS/MS.

The present study reports the degradation behaviour of a new prokinetic agent, Prucalopride succinate, under various stress conditions as per International Conference on Harmonization guidelines (ICH, Q1A (R2)). The investigation involved monitoring decomposition of the drug under hydrolytic (acidic, basic and neutral), oxidative, photolytic and thermal stress conditions followed by characterization of the degradation products (DPs) and process related impurities (IMPs). A rapid, precise, accurate and robust reverse phase high performance liquid chromatography (RP-HPLC) method has been developed involving mobile phase of 20mM ammonium bicarbonate buffer and acetonitrile: methanol (80:20v/v) on a Waters Xbridge-C8 (150mm×4.6mm i.d., 3.5µm) column using gradient elution. The drug was found to be degraded in hydrolytic (acidic) and oxidative conditions, whereas it was stable under basic and neutral hydrolytic, photolytic and thermal stress conditions. The method was extended to LC-ESI-QTOF-MS/MS for the structural characterization of DPs and process related IMPs. Structural characterization was carried out based on the generated molecular formula of DPs and its fragment ions. It has been observed that two major DPs were formed under each acid hydrolysis and oxidative stress conditions. The most probable mechanisms involved in the formation of DPs were also proposed. Finally, the method was validated in the term of specificity, linearity, accuracy, precision, and robustness as per ICH guidelines, Q2 (R1).

Forced degradation of fingolimod: effect of co-solvent and characterization of degradation products by UHPLC-Q-TOF-MS/MS and 1H NMR.

Fingolimod (FGL), an immunomodulator drug for treating multiple sclerosis, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per International Conference on Harmonization specified conditions. The drug showed extensive degradation under base hydrolysis, however, it was stable under all other conditions. A total of three degradation products (DPs) were observed. The chromatographic separation of the drug and its degradation products was achieved on a Fortis C18 (100×2.1mm, 1.7µm) column with a mobile phase composed of 0.1% formic acid (Solvent A) and acetonitrile (Solvent B) in gradient mode. All the DPs were identified and characterized by liquid chromatography-quadrupole time of flight-mass spectrometry (LC-Q-TOF-MS) in combination with accurate mass measurements. The major DP was isolated and characterized by Nuclear Magnetic resonance spectroscopy. This is a typical case of degradation where acetonitrile used as co-solvent in stress studies, reacts with FGL in base hydrolytic conditions to produce acetylated DPs. Hence, it can be suggested that acetonitrile is not preferable as a co-solvent for stress degradation of FGL. The developed UHPLC method was validated as per ICH guidelines.

Identification of hydrolytic and isomeric N-oxide degradants of vilazodone by on line LC-ESI-MS/MS and APCI-MS.

The present study reports the degradation behavior of a new antidepressant drug, vilazodone, under various stress conditions as per International Conference on Harmonization guidelines (ICH, Q1A(R2). The investigation involved monitoring decomposition of the drug under hydrolytic (acidic, basic and neutral), oxidative, photolytic and thermal stress conditions and identifying degradation products. A rapid, precise, accurate and robust ultra high performance liquid chromatography (UPLC) method has been developed on a Waters CSH Phenyl-Hexyl column (100 mm × 2.1 mm, 1.7 µm) using gradient elution of 10mM ammonium acetate buffer (pH 5.0) and acetonitrile as mobile phase. The drug was found to be degraded in hydrolytic (acidic and basic) and oxidative conditions, whereas it was stable under neutral hydrolytic, photolytic and thermal stress conditions. The method was extended to quadrupole time-of-flight mass spectrometry (QTOF-MS) for the structural characterization of degradation products. It has been observed that isomeric N-oxide degradation products were formed under oxidative stress condition. The exact location of N-oxidation in the drug was investigated using atmospheric pressure chemical ionization (APCI) due to the formation of characteristic fragment ions. These fragment ions resulted from Meisenheimer rearrangement owing to thermal energy activation at the vaporizer of APCI source. All degradation products were comprehensively characterized by UPLC-ESI-MS/MS and UPLC-APCI-MS experiments. The most probable mechanisms for the formation of degradation products have also been proposed. The method was validated in terms of specificity, linearity, accuracy, precision, and robustness as per ICH guidelines.

Structural characterization of alkaline and oxidative stressed degradation products of lurasidone using LC/ESI/QTOF/MS/MS.

A selective, accurate, precise and robust stability indicating liquid chromatography assay method was developed for the monitoring of a novel antipsychotic drug, lurasidone, in the presence of its degradation products (DPs). Also, we investigated degradation behavior of the drug under various stressed conditions such as hydrolytic (acidic, basic and neutral), oxidation, photolytic and thermal. The drug was found to be degraded under base hydrolytic and oxidative conditions, while it was stable in acid and neutral hydrolytic, photolytic and thermal conditions. The method showed adequate separation of lurasidone and its DPs on Xterra C18 (150 mm × 4.6 mm i.d., 3.5 µm) column using 20 mM ammonium formate (pH 3.0): acetonitrile as a mobile phase in gradient elution mode at a flow rate of 0.6 mL/min. This method was extended to liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC/ESI/QTOF/MS/MS) for structural characterization of DPs. A total of five DPs were characterized by LC/ESI/QTOF/MS/MS studies. Most probable mechanisms for the formation of DPs were proposed. The developed method was validated in terms of specificity, linearity, accuracy, precision, and robustness as per International Conference on Harmonization Guideline Q2 (R1).

LC-MS-MS Characterization of Forced Degradation Products of Fidarestat, a Novel Aldose Reductase Inhibitor: Development and Validation of a Stability-Indicating RP-HPLC Method.

An accurate, precise, robust and selective stability-indicating liquid chromatographic (LC) method has been developed for the monitoring of fidarestat in the presence of its forced degradants. The drug was subjected to hydrolysis (acid, alkali and neutral degradation), oxidation, photolysis and thermal stress conditions. The drug degraded significantly under hydrolytic (basic, acidic and neutral) and oxidative stress conditions, whereas it was found to be stable in photolytic and thermal conditions. The chromatographic separation was achieved on a Grace C18, (250 mm × 4.6 mm × 5 µm) column using gradient mobile phase system consisting of 10 mM of ammonium acetate buffer at pH 4 and acetonitrile at a flow rate of 1 mL/min with UV detection at 283 nm. The developed method was extended to liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS-MS) for characterization of all the degradation products. A total of five new degradation products were identified and characterized by LC-QTOF-MS-MS. The developed LC method was validated as per ICH guideline Q2 (R1). The proposed method was found to be successively applied for the quality control of fidarestat in bulk drug analysis.

Rapid structural characterization of in vivo and in vitro metabolites of tinoridine using UHPLC-QTOF-MS/MS and in silico toxicological screening of its metabolites.

Tinoridine is a nonsteroidal anti-inflammatory drug and also has potent radical scavenger and antiperoxidative activity. However, metabolism of tinoridine has not been thoroughly investigated. To identify in vivo metabolites, the drug was administered to Sprague-Dawley rats (n = 5) at a dose of 20 mg kg(-1), and blood, urine and feces were collected at different time points up to 24 h. In vitro metabolism was delved by incubating the drug with rat liver microsomes and human liver microsomes. The metabolites were enriched by optimized sample preparation involving protein precipitation using acetonitrile, followed by solid-phase extraction. Data processes were carried out using multiple mass defects filters to eliminate false-positive ions. A total of 11 metabolites have been identified in urine samples including hydroxyl, dealkylated, acetylated and glucuronide metabolites; among them, some were also observed in plasma and feces samples. Only two major metabolites were formed using liver microsomal incubations. These metabolites were also observed in vivo. All the 11 metabolites, which are hitherto unknown and novel, were characterized by using ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry in combination with accurate mass measurements. Finally, in silico toxicological screening of all metabolites was evaluated, and two metabolites were proposed to show a certain degree of lung or liver toxicity.

Characterization of forced degradation products of pazopanib hydrochloride by UHPLC-Q-TOF/MS and in silico toxicity prediction.

Pazopanib (PZ), an anti-cancer drug, was subjected to forced degradation under hydrolytic (acid, base and neutral), oxidative, photolytic and thermal stress conditions as per International Conference on Harmonization guidelines. A selective stability indicating validated method was developed using a Waters Acquity UPLC HSS T3 (100 × 2.1 mm, 1.7 µm) column in gradient mode with ammonium acetate buffer (10 mM, pH 5.0) and acetonitrile. PZ was found to degrade only in photolytic conditions to produce six transformation products (TPs). All the TPs were identified and characterized by liquid chromatography/atmospheric pressure chemical ionization-quadrupole-time of flight mass spectrometry experiments in combination with accurate mass measurements. Plausible mechanisms have been proposed for the formation of TPs. In silico toxicity was predicted using TOPKAT and DEREK softwares for all the TPs. The TP, N4-(2,3-dimethyl-2H-indazol-6-yl)-N4-methylpyrimidine-2,4-diamine, was found to be genotoxic, whereas all other TPs with sulfonamide moiety were hepatotoxic. The data reported here are expected to be of significance as this study foresees the formation of one potential genotoxic and five hepatotoxic degradation/transformation products.

Characterization of degradation products of ivabradine by LC-HR-MS/MS: a typical case of exhibition of different degradation behaviour in HCl and H2SO4 acid hydrolysis.

A validated stability-indicating HPLC method was established, and comprehensive stress testing of ivabradine, a cardiotonic drug, was carried out as per ICH guidelines. Ivabradine was subjected to acidic, basic and neutral hydrolysis, oxidation, photolysis and thermal stress conditions, and the resulting degradation products were investigated by LC-PDA and LC-HR-MS/MS. The drug was found to degrade in acid and base hydrolysis. An efficient and selective stability assay method was developed on Phenomenex Luna C18 (250 × 4.6 mm, 5.0 µm) column using ammonium formate (10 mM, pH 3.0) and acetonitrile as mobile phase at 30 °C in gradient elution mode. The flow rate was 0.7 ml/min and detection wavelength was 286 nm. A total of five degradation products (I-1 to I-5) were identified and characterized by LC-HR-MS/MS in combination with accurate mass measurements. The drug exhibited different degradation behaviour in HCl and H2SO4 hydrolysis conditions. It is a unique example where two of the five degradation products in HCl hydrolysis were absent in H2SO4 acid hydrolysis. The present study provides guidance to revise the stress test for the determination of inherent stability of drugs containing lactam moiety under hydrolytic conditions. Most probable mechanisms for the formation of degradation products have been proposed on the basis of a comparison of the fragmentation pattern of the drug and its degradation products. In silico toxicity revealed that the degradation products (I-2 to I-5) were found to be severe irritants in case of ocular irritancy. The analytical assay method was validated with respect to specificity, linearity, range, precision, accuracy and robustness.

Identification of forced degradation products of tamsulosin using liquid chromatography/electrospray ionization tandem mass spectrometry.

A rapid and gradient high-performance liquid chromatography combined with quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) method has been developed for the identification and structural characterization of stressed degradation products of tamsulosin. Tamsulosin, a selective α1-adrenoceptor antagonist, was subjected to forced degradation studies under hydrolytic (acid, base and neutral), oxidative, photolytic and thermal stress conditions as per ICH guidelines Q1A (R2). The drug degraded significantly under hydrolytic (base and neutral), thermal, oxidative and photolytic conditions, while it was stable to acid hydrolytic stress conditions. A total of twelve degradation products were formed and the chromatographic separation of the drug and its degradation products were achieved on a GRACE C-18 column (250mm×4.6mm, 5µm). All the degradants have been identified and characterized by LC/ESI-MS/MS and accurate mass measurements. To elucidate the structures of degradation products, fragmentation of the [M+H](+) ions of tamsulosin and its degradation products was studied by using LC-MS/MS experiments combined with accurate mass measurements. The product ions of all the protonated degradation products were compared with the product ions of protonated tamsulosin to assign most probable structures for the observed degradation products.

Characterization of forced degradation products of ketorolac tromethamine using LC/ESI/Q/TOF/MS/MS and in silico toxicity prediction.

Ketorolac, a nonsteroidal anti-inflammatory drug, was subjected to forced degradation studies as per International Conference on Harmonization guidelines. A simple, rapid, precise, and accurate high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (LC/ESI/Q/TOF/MS/MS) method has been developed for the identification and structural characterization of stressed degradation products of ketorolac. The drug was found to degrade in hydrolytic (acidic, basic, and neutral), photolytic (acidic, basic, and neutral solution), and thermal conditions, whereas the solid form of the drug was found to be stable under photolytic conditions. The method has shown adequate separation of ketorolac tromethamine and its degradation products on a Grace Smart C-18 (250 mm × 4.6 mm i.d., 5 µm) column using 20 mM ammonium formate (pH = 3.2): acetonitrile as a mobile phase in gradient elution mode at a flow rate of 1.0 ml/min. A total of nine degradation products were identified and characterized by LC/ESI/MS/MS. The most probable mechanisms for the formation of degradation products have been proposed on the basis of a comparison of the fragmentation of the [M + H](+) ions of ketorolac and its degradation products. In silico toxicity of the drug and degradation products was investigated by using topkat and derek softwares. The method was validated in terms of specificity, linearity, accuracy, precision, and robustness as per International Conference on Harmonization guidelines.