Identification of degradation products in stressed tablets of Rabeprazole sodium by HPLC‐hyphenated techniques

Three unknown impurities of Rabeprazole, a proton pump inhibitor, were formed in the formulated drug under the stress conditions, [40 °C/75% relative humidity (RH) for 6 months] with relative retention times (RRTs) 0.17, 0.22 and 0.28. The Impurity‐I (0.17 RRT) was isolated using preparative HPLC and characterized by NMR and MS. The other two impurities, Impurity‐II (RRT 0.22) and Impurity‐III (RRT 0.28) could not be isolated, hence they are characterized by HPLC‐hyphenated techniques, LC–NMR and high‐resolution LC–MS. On the basis of the spectral data, the Impurity‐I, Impurity‐II and Impurity‐III were characterized as 1‐(1H‐benzo[d]imidazol‐2‐yl)‐3‐methyl‐4‐oxo‐1,4‐dihydropyridine‐2‐carboxylic acid, 1H‐benzo [d] imidazole‐2‐sulfonic acid and 4‐(3‐methoxy propoxy)‐3‐methyl‐2‐pyridine carboxylic acid, respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of 2,6-dichlorobenzamide and its degradation products in water samples using solid-phase extraction followed by liquid chromatography–tandem mass spectrometry

An analytical method was developed for the determination of 2,6-dichlorobenzamide (BAM) and five degradation products thereof including 2-chlorobenzamide (OBAM), 2,6-dichlorobenzoic acid (DCBA), 2-chlorobenzoic acid (OBA), benzoic acid (BA) and benzamide (BAD) in water samples. Solid-phase extraction was combined with liquid chromatography coupled to tandem mass spectrometry using electrospray ionisation. Groundwater spiked at a concentration of 1.0 μg/L gave recoveries on day 1 between 91 and 102% (relative standard deviation: 2.2–26.5%) for OBAM, BAM, DCBA, BA and OBA, while BAD showed a somewhat lower recovery of 60% (relative standard deviation: 25%). Corresponding figures on day 3 gave recoveries of 97–110% (relative standard deviation: 3–22%) for OBAM, BAM, DCBA, BA and OBA, while BAD had a recovery of 51% (relative standard deviation: 4%). The final SPE-LC–MS/MS method had a LOD_Method of 0.009, 0.007, 0.010, 0.021, 0.253 and 0.170 μg/L groundwater for BAD, OBAM, BAM, DCBA, BA and OBA and a LOQ_Method of 0.030, 0.023, 0.035, 0.071, 0.842 and 0.565 μg/L groundwater in the same order of appearance. Analysis of three different Danish groundwaters confirmed the occurrence of BAM at levels exceeding the threshold value of 0.1 μg/L, while no degradation products were found above LOD_Method.     

Identification and characterization of stress degradation products of sumatriptan succinate by using LC/Q‐TOF‐ESI‐MS/MS and NMR: Toxicity evaluation of degradation products

Sumatriptan succinate, a selective 5‐HT1B receptor agonist, was subjected to forced degradation studies as per to International Conference on Harmonization‐specified conditions. The drug exclusively showed its degradation under basic, photolytic, and oxidative stress conditions, whereas it was found to be stable under acidic, thermal, and neutral conditions. Eight (DP‐1 to DP‐8) degradation products were identified and characterized by UPLC‐ESI/MS/MS experiments combined with accurate mass measurements. The effective chromatographic separation was achieved on Hibar Purospher STAR, C18 (250 × 4.6 mm, 5 μm) column using mobile phase consisting of 0.1% formic acid and methanol at a flow rate of 0.6 mL/minute in gradient elution method. It is noteworthy that 2 major degradation products DP‐3 and DP‐7 were isolated using preparative HPLC and characterized by advanced NMR experiments. The degradation pathway of the sumatriptan was established, which was duly justified by mechanistic explanation. In vitro cytotoxicity of isolated DPs was tested on normal human cells such as HEK 293 (embryonic kidney cells) and RWPE‐1 (normal prostate epithelial cells). This study revealed that they were nontoxic up to 100 μm concentration. Further, in silico toxicity of the drug and its degradation products was determined using ProTox‐II prediction tool. This study revealed that DP‐4 and DP‐8 are predicted for immune toxicity. Amine oxidase A and prostaglandin G/H synthase 1 are predicted as toxicity targets for DP‐3, DP‐4, and DP‐6 whereas DP‐1 and DP‐2 are predicted for amine oxidase A target.     

Newly identified degradation products of ceftiofur and cephapirin impact the analytical approach for quantitative analysis of kidney

This paper describes our research on the degradation of ceftiofur and cephapirin at physiological temperatures in kidney extract and in alkaline and acidic solution, conditions that regularly occur during sample preparation. Degradation products were identified using LC–ToF/MS, NMR and microbiological techniques. Additionally kinetics of the degradation processes were studied. A slight instability of cephapirin and desfuroylceftiofur was observed at elevated temperatures. Ceftiofur and cephapirin degraded immediately and completely in an alkaline environment, resulting in inactive degradation products. Ceftiofur and cephapirin also degraded immediately and completely in kidney extract resulting in both formerly reported metabolites as well as not previously reported products. Our research shows that conditions often occurring during the analysis of ceftiofur or cephapirin result in rapid degradation of both compounds. From this it is concluded that underestimation of the determined amounts of ceftiofur and cephapirin is likely to occur. Therefore, a new approach is needed for the analysis of both compounds newly identified degradation products.     

Stability-indicating HPLC method for the determination of nicardipine in capsules and spiked human plasma. Identification of degradation products using HPLC/MS

In this stability-indicating, reversed-phase high-performance liquid chromatographic method for nicardipine (NIC), forced degradation has been employed and the formed degradants were separated on a C18 (150 mm × 3.9 mm, 5 μm) analytical column using a mobile phase consisted of 70% methanol: acetic acid containing 0.01 M triethylamine with pH 4. The flow rate was 1.0 mL/min and the photodiode array detection wavelength was 353 nm. Forced degradation of the drug was carried out under acidic, basic, photolytic, and oxidative stress conditions. Chromatographic peak purity data indicated no co-eluting peaks with the main peaks. This method resulted in the detection of seven degradation products. Among these, two major degradation products from basic hydrolysis, one from oxidation by H₂O₂ and four from photolytic stress were identified by mass spectral data. A good linear response was achieved over the range of 0.5–40 μg/mL with a limit of detection (LOD) of 0.011 μg/mL and limit of quantification (LOQ) of 0.036 μg/mL. The suggested method was successfully applied for the analysis of NIC in its commercial capsules, with mean% recovery value of 100.11 ± 2.26%. The method was extended to the in vitro determination on NIC in spiked human plasma samples with mean% recovery of 99.04 ± 5.67%. The suggested method was utilized to investigate the kinetics of photolytic induced degradation.     

A strategy for the systematic development of a liquid chromatographic mass spectrometric screening method for polymer electrolyte membrane degradation products using isocratic and gradient phase optimized liquid chromatography

Within the scope of research for target and non-target LC–MS/MS analysis of membrane degradation products of polymer electrolyte membrane fuel cells, a systematic method development for the separation of structurally similar compounds was performed by phase optimized liquid chromatography. Five different stationary phases with different selectivities were used. Isocratic separation for 4-hydroxybenzoic acid, isophthalic acid, terephthalic acid, 4-hydroxybenzaldehyde and 4-formylbenzoic acid was achieved on a C18 and a Phenyl phase. Using the PRISMA model the separation efficiency was optimized. This was achieved on a serially connected mixed stationary phase composed of 30 mm C18, 150 mm Phenyl and 60 mm C30. For the LC–MS screening of unknown degradation products from polymer electrolyte membranes in the product water of a fuel cell, a solvent gradient is mandatory for less polar or later eluting compounds. By means of 4-mercaptobenzoic acid it could be shown that a solvent gradient can be applied in order to elute later eluting compounds in a short time. The adaptability of this method for the qualitative analysis by target and non-target LC–MS/MS screening has been shown by means of 4-hydroxybenzoic acid. The combination of solvent gradient and isocratic conditions makes this approach attractive for the purpose of a screening method for known and unknown analytes in a water sample.     

Identification of degraded products of aldicarb due to the catalytic behavior of titanium dioxide/polyacrylonitrile nanofiber

Photocatalytic properties of fibers containing TiO₂ nanoparticles were explored for use as a self-decontaminating material using degradation of the pesticide aldicarb as the model toxin. During the analysis of the aldicarb treated sample by liquid chromatography (LC) with diode array detector (DAD), an unidentified peak was found at relative retention time (RT) 3.9 min when compared to aldicarb and major metabolites, aldicarb sulfoxide, and aldicarb sulfone. An analytical method was developed to confirm and identify this degradation product. LC–APCI/MS techniques were used first to analyze molecular ions and major fragments comparing retention times and spectra with those of known standards. FTIR and LC–MS/MS techniques were used to confirm the identity of the degradation product as 2-propenal, 2-methyl-, O-[(methylamino)carbonyl]oxime.     

Elucidation of stress‐induced degradation products of mangiferin: Method development and validation

The degradation behavior of mangiferin, under various ICH Q1A(R2) recommended stress conditions, was studied using an isocratic elution with mobile phase (pH 2.4), composed of acetonitrile and 1% orthophosphoric acid (12:88 v/v) at a flow rate of 1.0 mL/min, with λ _max 262 nm. It was suitably adapted for LC–MS studies by replacing with 1% acetic acid (ACN–1% acetic acid; 18:82) and the pH was adjusted to 3.0. Extensive degradation was found to occur during alkaline medium stress studies at 2.31 min of retention time at λ _max of 235 nm. The mass spectrum of mangiferin, 3 h after treatment with 0.1 M NaOH, clearly shows the rupture of the tricyclic ring, indicating that a fragment at m /z − 269 was formed. Furthermore, the results were supported by nuclear magnetic resonance as well. However, no degradation was observed in other stress conditions.     

Determination of amitraz and its degradation products and monitoring degradation process in quince and cucumber

Amitraz is a non-systemic acaracide and insecticide. Current maximum residue limits for amitraz are stated as ‘Amitraz including the metabolites containing the 2,4-dimethylaniline moiety’. Therefore, determination of amitraz and its all degradation products are important. In this study, we develop a gas chromatography/mass spectrometry (GC/MS) method for determination of amitraz and its degradation products 2,4 dimethylaniline (DMA), 2,4 dimethylformamidine (DMF) and N-(2,4-dimethyl phenyl)-N’-methylformamidine (DMPF) in cucumber and quince. The mechanism of the degradation process was monitored at different temperatures. Amitraz and its degradation products were extracted using the QuEChERS method. To determine amitraz and its degradation products, we used GC/MS. Quantification was carried out by using selected ion monitoring, and total ion chromatogram was used to monitor additional degradation products. The method was validated by studying linearity, limit of detection (LOD) and limit of quantification (LOQ), recovery and precision. The mechanism of the degradation process was monitored at different temperatures. Degradation of amitraz mainly to three degradation products, namely DMA, DMF and DMPF, increased with temperature. Besides these three main degradation products, two other new degradation products were detected.     

Characterization of human hair melanin and its degradation products by means of magnetic resonance techniques

Melanin granules (MGs) have been extracted from human Chinese black hairs by either acid hydrolysis (CH-type MGs) or enzymatic digestion (CP-type MGs), and their chemical structure investigated at the solid state by means of (13)C cross polarization magic angle spinning (CPMAS NMR) and EPR spectroscopy. Both types of MGs contain a large amount of protein that is tightly bound to the true melanin polymer, with CP-type MGs having a larger protein content than CH-type ones. Moreover, MGs may also contain variable amounts of lipid-like material. A high amount of paramagnetic metals is detected by EPR in CP-type MGs, in particular Fe(III). Iron can be bound in two chemical forms: as isolated high spin Fe(III) ions with rhombic symmetry and as small oxy-hydroxy Fe(III) aggregates. Iron is poorly available to chelators. CH-type MGs contain much fewer metals. CP-type MGs have then been subjected to partial bleaching by hydrogen peroxide in ammonia, yielding a residual solid, called residual oxidized melanin (ROM) and a soluble but still pigmented fraction called melanin free acid (MFA). MFA can be isolated by precipitation at acidic pH. The (13)C-CPMAS NMR and EPR spectra of these derivatives indicated that ROM has a structure very similar to that of parent MGs, whereas MFA shows a decrease of the protein content with respect to the melanin and a decreased amount of bound iron. Thus, the oxidative degradation of CP-type MGs is a process not involving the bulk of MGs, but rather it proceeds from the solvent-exposed outer parts to the interior.     

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. Copyright © 2014 John Wiley & Sons, Ltd.     

Residue analysis of tebufenozide and indoxacarb in chicken muscle,milk, egg and aquatic animal products using liquid chromatography–tandem mass spectrometry

We developed an analytical method using liquid–liquid extraction (LLE) and liquid chromatography–tandem mass spectrometry (LC‐MS/MS) to detect and quantify tebufenozide (TEB) and indoxacarb (IND) residues in animal and aquatic products (chicken muscle, milk, egg, eel, flatfish, and shrimp). The target compounds were extracted using 1% acetic acid (0.1% acetic acid for egg only) in acetonitrile and purified using n‐hexane. The analytes were separated on a Gemini‐NX C₁₈ column using (a) distilled water with 0.1% formic acid and 5 mm ammonium acetate and (b) methanol with 0.1% formic acid as the mobile phase. All six‐point matrix‐matched calibration curves showed good linearity with coefficients of determination (R²) ≥0.9864 over a concentration range of 5–50 μg/kg. Intra‐ and inter‐day accuracy was expressed as the recovery rate at three spiking levels and ranged between 73.22 and 114.93% in all matrices, with a relative standard deviation (RSD, corresponding to precision) ≤13.87%. The limits of quantification (LOQ) of all target analytes ranged from 2 to 20 μg/kg, which were substantially lower than the maximum residue limits (MRLs) specified by the regulatory agencies of different countries. All samples were collected from different markets in Seoul, Republic of Korea, and tested negative for tebufenozide and indoxacarb residues. These results show that the method developed is robust and may be a promising tool to detect trace levels of the target analytes in animal products.     

Stability of doripenem in reconstituted solution – thermal and oxidative decomposition kinetics and degradation products by LC–MS

A ultra‐fast liquid chromatography method applied to quantitation of doripenem in powder for injection was validated. Validation parameters were assayed and a rapid analysis was established by a reversed‐phase system comprising a C₁₈ column endcapped (50 × 4.0 mm, 2.0 μm), mobile phase consisting of phosphoric acid 0.01% (pH 3.8) and acetonitrile (98:02, v /v) and a flow rate of 0.4 mL min⁻¹. Drug stability was studied through submission to forced conditions, allowing the major degradation products to be detected and the kinetics parameters to be established. Thermal and oxidative degradation were determined, and indicated a kinetic decomposition following first and second order, respectively. The main degradation products were identified by LC–MS analysis, and the results were evaluated in order to suggest the chemical structures corresponding to respective masses and fragmentations. Six compounds were identified, with m/z 411, 427, 437, 634, 650 and 664. All of them resulted from cleavage of β ‐lactam ring and alcoholic chain and/or dimerization. These experimental results provide valuable information about the stability of doripenem reconstituted solution and procedures for its handling and storage.     

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 m m , 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. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification and characterization of stressed degradation products of metoprolol using LC/Q-TOF-ESI-MS/MS and MS(n) experiments

A rapid, specific and reliable isocratic 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 and validated for the identification and characterization of stressed degradation products of metoprolol. Metoprolol, an anti-hypertensive drug, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per ICH-specified conditions. The drug showed extensive degradation under oxidative and hydrolysis (acid and base) stress conditions. However, it was stable to thermal, neutral and photolysis stress conditions. A total of 14 degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on a C(18) column (4.6 × 250 mm, 5 μm). To characterize degradation products, initially the mass spectral fragmentation pathway of the drug was established with the help of MS/MS, MS(n) and accurate mass measurements. Similarly, fragmentation pattern and accurate masses of the degradation products were established by subjecting them to LC-MS/QTOF analysis. Structure elucidation of degradation products was achieved by comparing their fragmentation pattern with that of the drug. The degradation products DP(2) (m/z 153) and DP(14) (m/z 236) were matched with impurity B, listed in European Pharmacopoeia and British Pharmacopoeia, and impurity I, respectively. The LC-MS method was validated with respect to specificity, linearity, accuracy and precision.     

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. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of major degradation products of an adenosine A2A receptor antagonist under stressed conditions by LC‐MS and FT tandem MS analysis

Parkinson's disease (PD) is a very serious neurological disorder, and current methods of treatment fail to achieve long‐term control. SCH 420814 is a potent, selective and orally active adenosine A_2A receptor antagonist discovered by Schering‐Plough. Stability testing provides evidence of the quality of a bulk drug when exposed to the influence of environmental factors. Understanding the drug degradation profiles is critical to the safety and potency assessment of the drug candidate for clinical trials. As a result, identification of degradation products has taken an important role in drug development process. In this study, a rapid and sensitive method was developed for the structural determination of the degradation products of SCH 420814 formed under different forced conditions. The study utilizes a combination of liquid chromatography–tandem‐mass spectrometry (LC‐MS/MS) and Fourier Transform (FT) MS techniques to obtain complementary information for structure elucidation of the unknowns. This combination approach has significant impact on degradation product identification. A total of ten degradation products of SCH 420814 were characterized using the developed method. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of chlormequat and mepiquat by hydrophilic interaction chromatography coupled to tandem mass spectrometry in food samples

In this work a LC–MS/MS method for the determination of two quaternary ammonium growth regulators (chlormequat and mepiquat) in food is reported. The separation was based on hydrophilic interaction liquid chromatography (HILIC) without the use of ion-pair reagents. A gradient elution of acetonitrile and formic acid/ammonium formate buffer from 60 to 40% acetonitrile was enough to achieve a resolution >1.5 in less than 4.0 min. The HILIC system was coupled to a triple quadrupole mass spectrometer equipped with a heated electrospray probe (H-ESI) providing sub-pg LODs in SRM mode. A straightforward sample treatment (SPE C18 clean-up) was enough to provide MLODs at low ppb levels when analysing a range of food samples that covered different kinds of matrices such as fresh fruit, vegetables, fruit juices, baby food, bread, coffee and beer. Chlormequat was found in seven samples (0.8–126 ng/g) but mepiquat was only detected in bread and coffee samples (0.9–166 ng/g).     

Studies on thermal degradation of 1-4 and 1-2 polybutadienes in inert atmosphere

The degradation of 1,2 and 1,4 polybutadienes by heating in inert atmosphere was characterized by FTIR, NMR and TGA/TCT/GC/MS for the volatile organic compounds. Two distinct mass change stages in the thermogravimetric analysis indicated different temperature ranges of degradation. Below 300 °C, the predominant reactions depend on the chemical structure of PBs. Thus, M1 and M2 formation by heating 1,4-PB at 260 °C allows us to prove crosslinking reaction. On the other hand, heating 1,2-PB at 260 °C leads to decahydronaphthalene and methyl formation by cycloaddition and rearrangement. At about 300 °C, only radical scission occurs for 1,2-PB whereas Diels Alder and proton transfer mechanisms are described for 1,4-PB. The products of reaction are respectively conjugated diene, cyclic and linear unsaturated compounds sometimes with methyl groups. Above 400 °C, the main process is aromatisation for both PBs.     

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).