Development of stability-indicating method for separation and characterization of benidipine forced degradation products using LC–MS/MS

The present study describes forced degradation of benidipine (BEN) as per  Q1A (R2) and Q1B guidelines of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. BEN degraded under hydrolysis (neutral, acidic, and alkaline), hydrogen peroxide induced oxidation, and UV light mediated photolytic degradation. A total of 14 degradation products (DPs) were found in all degradation studies, comprising 4 hydrolytic DPs, 8 oxidative DPs, and 4 photolytic DPs. A selective stability-indicating method was developed using an XBridge BEH C18 column with gradient elution program consisting of ammonium acetate (10 mM, 4.8 pH, acetic acid) and acetonitrile. The flow rate was maintained at 1 ml min⁻¹. All DPs were separated well using the developed HPLC method and were characterized using LC–MS/MS data. As this method is effective in identifying and separating BEN and its DPs with sufficient resolution, it can be used in laboratories for quality control of drugs in daily routine analysis and stability studies.     

Study of degradation behavior of besifloxacin,characterization of its degradation products by LC–ESI–QTOF–MS and their in silico toxicity prediction

Knowledge and understanding of the stability profile of a drug is important as it affects its safety and efficacy. In the present work, besifloxacin, a new, fourth‐generation fluoroquinolone antibiotic, was subjected to different forced‐degradation conditions as per International Conference on Harmonization (ICH) guidelines such as hydrolysis (acid, base and neutral), oxidation, thermal and photolysis. The drug degraded under acidic, basic, oxidative and photolytic conditions while it was found to be stable under dry heat and neutral hydrolytic conditions. In total, five degradation products (DPs) were formed under different conditions—DP1 and DP2 (photolysis), DP3 (oxidation), DP4 (acidic), DP3 and DP5 (basic). The chromatographic separation of besifloxacin and its degradation products was achieved on a Sunfire C₁₈ (250 mm × 4.6 mm, 5 μm) column with 0.1% aqueous formic acid–acetonitrile as a mobile phase. The gradient RP‐HPLC method was developed and validated as per ICH guidelines. The degradation products were characterized with the help of LC–ESI–QTOF mass spectrometric studies and the most likely degradation pathway of the drug was proposed. In silico toxicity assessment of the drug and its degradation products was carried out, which indicated that DP3 and DP4 carry a mutagenicity alert.     

Comparison of Two Stability-Indicating Chromatographic Methods for the Determination of Mirabegron in Presence of Its Degradation Product

Mirabegron is a novel β₃-adrenoceptor agonist containing an amide group. It was subjected to stress conditions of acidic and alkaline hydrolyses. The hydrolytic degradation product was isolated and its structure was confirmed using mass and IR spectrometry. Two stability-indicating chromatographic methods have been proposed for the determination of mirabegron. TLC method was applied using silica gel as stationary phase and chloroform–methanol–ammonia (9.0:1.0:0.1 by volume) as the mobile phase, and chromatograms were scanned at 250 nm. Accurate determination of the drug was achieved over the concentration range of 2–12 μg per band. In addition, an isocratic HPLC method was developed on Agilent C18 column (150 mm × 4.5 mm I.D., particle size 5 µm) using ethanol-phosphate buffer pH 2.5 (30:70, by volume) as a mobile phase with flow rate of 1 mL min^?1.The intact drug was detected at 250 nm with running time less than 5 min. Mirabegron was determined accurately in a concentration range of 1–25 µg mL^?1. The proposed chromatographic methods were applied successfully for the assay of mirabegron in pharmaceutical dosage form and both methods were validated as per the International Conference on Harmonization guidelines and statistically compared with a reported gradient HPLC method.     

Characterization of stress degradation products of benazepril by using sophisticated hyphenated techniques

Benazepril, an anti-hypertensive drug, was subjected to forced degradation studies. The drug was unstable under hydrolytic conditions, yielding benazeprilat, which is a known major degradation product (DP) and an active metabolite. It also underwent photochemical degradation in acid and neutral pH conditions, resulting in multiple minor DPs. The products were separated on a reversed phase (C18) column in a gradient mode, and subjected to LC–MS and LC–NMR studies. Initially, comprehensive mass fragmentation pathway of the drug was established through support of high resolution mass spectrometric (HR-MS) and multi stage tandem mass spectrometric (MSⁿ) data. The DPs were also subjected to LC–MS/TOF studies to obtain their accurate masses. Along with, on-line H/D exchange data were obtained to ascertain the number of exchangeable hydrogens in each molecule. LC–¹H NMR and LC–2DNMR data were additionally acquired in a fraction loop mode. The whole information was successfully employed for the characterization of all the DPs. A complete degradation pathway of the drug was also established.     

Structural characterization of forced degradation products of empagliflozin by high resolution mass spectrometry

Eleven unique degradation products (DPs) of empagliflozin (EGF), a sodium glucose cotransporter (SGLT) 2 inhibitor have been reported for the first time. These DPs were generated as per International Conference on Harmonization (ICH) guidelines Q1 (R2) using stress conditions such as acid, base hydrolysis and oxidative environment. The sum of 12 DPs have been successfully resolved by Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) method involving Eclipse plus C₁₈ RRHD (2.1?×?50?mm, 1.8µm) column and mobile phase comprising of water with 0.1% formic acid and methanol in 1:1 quotient. Structure of DPs and the degradation pathways have been proposed on the basis of accurate mass and MS/MS fragmentation pattern acquired through Liquid Chromatography-Electrospray Ionization Quadrupole Time of Flight Mass Spectrometer (LC-ESI-QTOF-MS). It has been observed that maximum DPs were formed during acid hydrolysis and the major ones were (2S,3R,4R,5S,6R)-2-(4-chloro-3-(4-(((S)-1,4-dihydroxybutan-2-yl)oxy)benzyl)phenyl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (DP1) and (2S,3R,4R,5S,6R)-2-(4-chloro-3-(4-hydroxybenzyl)phenyl)-6-(hydroxymethyl) tetrahydro-2H-pyran-3,4,5-triol (DP2) generated due to ring opening of tetrahydrofuran moiety and elimination of tetrahydrofuran ring respectively. The proposed structures of DPs and their associated pathways will be essential for optimization of manufacturing and quality control parameters of EGF.     

Study of Forced Degradation Behaviour of Brinzolamide Using LC–ESI–Q-TOF and In Silico Toxicity Prediction

Brinzolamide (BZA) is a topical ophthalmic drug which is generally used to lower the intraocular pressure during glaucoma. It was subjected to forced degradation studies under hydrolytic (acidic, basic), oxidative, photolytic and thermal stress conditions; the drug degraded significantly in hydrolytic and oxidative conditions, leading to the formation of seven degradation products in total. It was stable on exposure to light and dry heat in the solid state. An ultra-performance liquid chromatography (UPLC) method was developed on a Waters CSH phenyl hexyl column (100 × 2.1 mm, 1.7 µm), using gradient elution of 0.1 % formic acid and methanol as mobile phase. The method was extended to quadrupole time-of-flight mass spectrometry (Q-TOF–MS) for the structural characterisation. All degradation products were comprehensively characterised by UHPLC–ESI/MS/MS experiments. The most probable mechanisms for the formation of degradation products were also proposed. In silico toxicity of the drug and its degradation products was determined using TOPKAT toxicity prediction software.

     

Characterization of Nineteen Impurities in Roxithromycin by HPLC/TOF and Ion Trap Mass Spectrometry

Nineteen impurities in roxithromycin drug substance made in China were separated and identified by HPLC–MSⁿ (TOF and TRAP) for the further improvement of official monographs in Pharmacopoeias. The fragmentation patterns and structural assignment of these impurities were studied. The column was Shim VP-ODS (250 × 4.6 mm, 5 μm). The mobile phase was 10 m mol L^?1 ammonium acetate and 0.1 % formic acid aqueous solution-acetonitrile (62.5:37.5). In positive mode, full scan LC–MS was first performed to obtain the m/z value of the protonated molecules and formulas of all detected peaks on Agilent 6538Q TOF high resolution mass spectrometer. LC–MS-MS and LC–MS-MS–MS were then carried out on the compounds of interest on AB SCIEX 4000 Q TRAP? composite triple quadrupole/linear ion trap tandem mass spectrometer. The complete fragmentation patterns of nineteen impurities were studied and used to obtain information about the structures of these impurities. The structures of nineteen impurities in roxithromycin drug substance were deduced based on the HPLC–MSⁿ data, in which nine impurities were novel impurities.     

Characterization of Nineteen Impurities in Roxithromycin by HPLC/TOF and Ion Trap Mass Spectrometry

Nineteen impurities in roxithromycin drug substance made in China were separated and identified by HPLC–MSⁿ (TOF and TRAP) for the further improvement of official monographs in Pharmacopoeias. The fragmentation patterns and structural assignment of these impurities were studied. The column was Shim VP-ODS (250 × 4.6 mm, 5 μm). The mobile phase was 10 m mol L⁻¹ ammonium acetate and 0.1 % formic acid aqueous solution-acetonitrile (62.5:37.5). In positive mode, full scan LC–MS was first performed to obtain the m/z value of the protonated molecules and formulas of all detected peaks on Agilent 6538Q TOF high resolution mass spectrometer. LC–MS-MS and LC–MS-MS–MS were then carried out on the compounds of interest on AB SCIEX 4000 Q TRAP™ composite triple quadrupole/linear ion trap tandem mass spectrometer. The complete fragmentation patterns of nineteen impurities were studied and used to obtain information about the structures of these impurities. The structures of nineteen impurities in roxithromycin drug substance were deduced based on the HPLC–MSⁿ data, in which nine impurities were novel impurities.

     

LC–MS/MS and NMR characterization of forced degradation products of mirabegron

A rapid, precise, and reliable liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed for the characterization of stressed degradation products of mirabegron. It is used in the treatment of overactive bladder and administered to treat urinary symptoms such as urgency or frequency and incontinence. It also works by relaxing the muscles around bladder.

Mirabegron was subjected to hydrolysis (acidic, alkaline, and neutral) and peroxidation, as per ICH-specified conditions. The drug showed degradation under stress conditions. However, it was stable to neutral conditions. A total of seven degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on X-TerraRP-8 (250 mm × 4.6 mm, i.d., 5 µm) column using 0.01 M ammonium acetate as mobile phase-A and 60:40 ratio of acetonitrile (ACN):water as mobile phase-B. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. Probable possible structures were drawn based on parent and daughter molecular ions. One peroxide degradant impurity was isolated using preparative LC and characterized using liquid chromatography–mass spectrometry and NMR data.     

Experimental Design Approach for Selective Separation of Vilazodone HCl and Its Degradants by LC-PDA and Characterization of Major Degradants by LC/QTOF–MS/MS

A selective, rapid, accurate and precise stability-indicating RP-HPLC method was developed for monitoring vilazodone in the presence of its degradation products (DPs) in API and pharmaceutical dosages. Chromatographic separation was achieved using a Grace phenyl C₁₈ column (250 × 4.6 mm, 5 µm particle size) at a flow rate of 1.0 mL min^?1 with a linear gradient elution of ammonium acetate buffer (10 mM, pH 5.0 adjusted by acetic acid) with acetonitrile as the mobile phase. Detection was performed at 240 nm. The chromatographic conditions were optimized by the design of experiments to obtain the best possible separation with a minimum number of trials. A face-centered central composite design (three levels for each factor) was employed for optimization, and an interaction of the independent variables (pH of mobile phase, column temperature and  % of organic modifier) on the resolution of critical pairs was studied. The drug was subjected to the stress conditions of hydrolytic (acid, base and neutral), oxidative, thermal and photolytic degradation. It was found to be degraded significantly in hydrolytic (basic and acid) and oxidative conditions, while it was stable in neutral hydrolytic, thermal and photolytic conditions. LC-QTOF/MS/MS studies were carried out to characterize the major DPs. The method was fully validated for selectivity, linearity, accuracy, precision and robustness in compliance with ICH guideline Q2 (R1).     

Forced Degradation Study on Gliclazide and Application of Validated Stability-Indicating HPLC-UV Method in Stability Testing of Gliclazide Tablets

Forced degradation study on gliclazide was conducted under the conditions of hydrolysis, oxidation, dry heat and photolysis and an isocratic stability-indicating HPLC-UV method was developed and validated. All the seven degradation products (I–VII) formed under different conditions were optimally resolved on a C₁₈ column with mobile phase composed of 40% acetonitrile and 60% ammonium acetate solution (0.025 M, pH 3.5) at a flow rate of 0.25 mL min^?1 using 235 nm as detection wavelength. The method was linear between 5–500 μg mL^?1 drug concentrations. The %RSD of intra- and inter-day precision studies was <1 and <2% respectively. Excellent recoveries (99.81–100.97%) proved the method sufficiently accurate. Each peak resolved always with a resolution of >1.90 indicating the method to be rugged enough. The method was used to study the drug degradation behaviour under the forced conditions. Four degradation products (I–IV) were formed in 0.1 N HCl and water whereas only I and III were formed in 3% H₂O₂. Two new products V and VI in addition to I, III and IV were formed in 0.1 N NaOH. The drug was stable to thermal and photolytic decomposition. The degradation behaviour in water and 0.1 N NaOH was similar under dark and light conditions but a new product VII was formed in 0.01 N HCl in light. In general, the rate of degradation was accelerated by the light. The method was applied successfully in stability testing of gliclazide tablets.     

Kinetic study of the degradation of forskolin in aqueous systems by stability-indicating HPLC method and identification of its degradation products

The degradation kinetics of forskolin in aqueous solution was investigated qualitatively and quantitatively. Two degradation products were isolated and identified as isoforskolin and forskolin D by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. A stability-indicating high-performance liquid chromatography (HPLC) method was developed and validated for the quantification of forskolin and its degradation products. Chromatographic separation was performed on a Luna C₁₈ column with acetonitrile–water (65:35, v/v) as the mobile phase. The flow rate was kept at 1 mL/min, and the detection wavelength was 210 nm. The kinetic study of forskolin was carried out in aqueous solutions of pH 1.5–8.5 at 37, 50, 65, and 80°C. The degradation rate of forskolin increases with increasing temperature. Forskolin is relatively stable in the pH range 3.5–6.5, but its stability decreases when the pH is outside this range. In the pH range 6.5–8.5, the forskolin degradation follows pseudo-first-order kinetics. Based on the structural identification and quantitative analysis of the degradation products, a possible pathway for forskolin degradation is proposed. Forskolin can be converted to isoforskolin rapidly, and both forskolin and isoforskolin can further decompose to forskolin D.     

Stability-Indicating TLC Method for the Determination of Dutasteride in Pharmaceutical Dosage Forms

A simple, sensitive, selective, precise and stability-indicating thin-layer chromatographic method for determination of dutasteride both as a bulk drug and as pharmaceutical tablets was developed and validated as per the International Conference on Harmonization guidelines. The method employed thin-layer chromatography aluminium plates precoated with silica gel 60F₂₅₄ as the stationary phase and the mobile phase consisted of acetonitrile:methanol:dichloromethane in the ratio of 2.0:1.0:2.0, v/v/v. This solvent system was found to give compact spots for dutasteride (R _f value of 0.64 ± 0.02). Densitometric analysis of dutasteride was carried out in the absorbance mode at 244 nm. The linear regression analysis data for the calibration plots showed good linear relationship with r = 0.9943 with respect to peak area in the concentration range of 100–600 ng per band. The method was validated for precision, accuracy, ruggedness and recovery. The limits of detection and quantitation were 7.54 and 22.85 ng per band, respectively. Dutasteride was subjected to acid and alkali hydrolysis, oxidation, photo degradation, dry heat and wet heat treatment. The drug undergoes degradation under acidic, basic conditions, photolytic, oxidative and upon wet and dry heat treatment. The degraded products were well separated from the pure drug. The statistical analysis proves that the developed method for quantification of dutasteride as bulk drug and from pharmaceutical tablets is reproducible and selective. As the method could effectively separate the drug from its degradation products, it can be employed as stability-indicating.     

Stress Degradation Studies of Anagliptin,Development of Validated Stability Indicating Method,Degradation Kinetics Study,Identification and Isolation of Degradation Products

A gradient-specific stability indicating HPLC method was developed and validated for the determination of the antidiabetic agent anagliptin in laboratory mixtures. Reversed-phase chromatography was performed using a Shimadzu LC-20 AD pump (binary), Shimadzu PDA M-20A diode array detector, and Waters Symmetry C-18 column (150?×?4.6 mm, 3.5 µm) maintained at a column oven temperature of 40 °C with UV detection at 247 nm. A gradient program was run at flow rate of 1 mL min^?1. Mobile phase A consisted of a mixture of acetate buffer(10 mm) pH 5/methanol/acetonitrile in the ratio of 90:5:5. Mobile phase B consisted of a mixture of acetate buffer (10 mm) pH 5/methanol/acetonitrile in the ratio of 50:25:25. The method was validated according International Conference of Harmonization (ICH) guidelines. Linearity was observed in the concentration range of 10–120 µg/mL with regression coefficient r²(0.999). The LOD was found to be 7.8 µg/mL and LOQ was found to be 22.68 µg/mL. Anagliptin was subjected to stresses such as acidic, alkali, oxidation, photolysis, and thermal conditions. The proposed method was validated as per ICH guidelines and was found to be accurate, precise, and specific. The drug showed significant degradation in alkaline and oxidative conditions. Alkaline and oxidative degradation followed first-order kinetics. Degradation rate constant and half-lives were determined. Degradation products in alkaline and oxidative conditions were identified by LC–MS. One major degradation product was isolated from each condition by preparative HPLC. These degradation products were characterized by ¹H NMR, ¹³C NMR, DEPT, D₂O exchange, MS/MS, HRMS, and IR techniques. From the spectral data the alkaline degradation product was characterized as 1-{2-[1-(2-methylpyrazolo[1,5-a]pyrimidine-6-carboxamido)-methyl-propan-2-yl-amino]acetyl}pyrrolidine-2-carboxamide. The oxidative degradation product was characterized as N-[2-({2-[(2S)-2-cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo-[1,5-a]pyrimidine-N-oxido-6-carboxamide.     

Stability-Indicating HPTLC Method for the Determination of Tamsulosin in Pharmaceutical Dosage Forms

A simple, sensitive, selective, precise and stability indicating high-performance thin-layer chromatographic method was developed for the determination of tamsulosin (TAM) in bulk and tablet formulation. Validation was carried out in compliance with International Conference on Harmonization guidelines. The method employed thin-layer chromatography aluminium plates pre-coated with silica gel 60F₂₅₄ as the stationary phase and the mobile phase consisted of acetonitrile/methanol/dichloromethane (2.0: 1.0: 2.0, v/v/v). This solvent system was found to give compact spots for tamsulosin (R _f = 0.27 ± 0.02). Densitometric analysis of TAM was carried out in the absorbance mode at 286 nm. Linear regression analysis showed good linearity (r ² = 0.9993) with respect to peak area in the concentration range of 300–800 ng per band. The method was validated for precision, accuracy, ruggedness and recovery. Limits of detection and quantitation were 8.49 and 25.72 ng per band, respectively. TAM was subjected to acid and alkali hydrolysis, oxidation, photo degradation, dry heat and wet heat treatment. The drug underwent degradation under acidic, basic and photolytic conditions. The degraded products were well separated from the pure drug. Statistical analysis proved that the developed method, used for quantification of TAM as a bulk drug and present in pharmaceutical tablets, was reproducible and selective.

     

Stability-Indicating HPTLC Method for the Determination of Tamsulosin in Pharmaceutical Dosage Forms

A simple, sensitive, selective, precise and stability indicating high-performance thin-layer chromatographic method was developed for the determination of tamsulosin (TAM) in bulk and tablet formulation. Validation was carried out in compliance with International Conference on Harmonization guidelines. The method employed thin-layer chromatography aluminium plates pre-coated with silica gel 60F₂₅₄ as the stationary phase and the mobile phase consisted of acetonitrile/methanol/dichloromethane (2.0: 1.0: 2.0, v/v/v). This solvent system was found to give compact spots for tamsulosin (R _f = 0.27 ± 0.02). Densitometric analysis of TAM was carried out in the absorbance mode at 286 nm. Linear regression analysis showed good linearity (r ² = 0.9993) with respect to peak area in the concentration range of 300–800 ng per band. The method was validated for precision, accuracy, ruggedness and recovery. Limits of detection and quantitation were 8.49 and 25.72 ng per band, respectively. TAM was subjected to acid and alkali hydrolysis, oxidation, photo degradation, dry heat and wet heat treatment. The drug underwent degradation under acidic, basic and photolytic conditions. The degraded products were well separated from the pure drug. Statistical analysis proved that the developed method, used for quantification of TAM as a bulk drug and present in pharmaceutical tablets, was reproducible and selective.     

Ultra-High Performance Method on Superficially Porous Stationary Phase for the Determination of Related Substances in Pitavastatin Calcium by HPLC

A simple reverse-phase method for the selective quantification of pitavastatin calcium (PIT) and its related substances was developed. The method demonstrated an excellent separation between PIT and each of 15 impurities (including its isomers and degradants) within a short run time of 12 min by HPLC. A rapid resolution similar to that of UHPLC was achieved using high flow rate on superficially porous C18 stationary phase. A synergistic combination of quality by design approach and use of a superficially porous column delivered a HPLC method with ultra-high performance. Forced degradation studies proved the method to be highly specific (mass balance > 98 %) and the structures of major degradation products were proposed based on LC–MS analysis. The results of validation proved the method to be highly precise (%RSD < 4), accurate (recoveries in range of 100 ± 7 %), linear (r ² > 0.999) and sensitive (LOQ ≤ 0.02 % and LOD ≤ 0.005 %) for all the impurities and drug. Use of multivariate analysis helped to incorporate high robustness in the method. The method is valuable for quantification of PIT and its related substances in both drug substance and oral solid dosage form.

     

Development and Validation of Liquid Chromatography–Tandem Mass Spectrometry for Simultaneous Determination of the Four Active Components of Danhong Freeze‐dried Powder Injection in Human Plasma

Abstract

A liquid chromatography‐tandem mass spectrometry was developed and validated for the simultaneous determination of protocatechuic aldehyde, danshensu, salvianolic acid B, and hydroxysafflor yellow A, with rutin as internal standard. Electrospray ionization patterns and efficiency of the analytes, along with their fragmentation, were investigated to achieve sensitive electrospray tandem mass spectrometry (ESI‐MS/MS) detection. Plasma samples were extracted by solid‐phase extraction columns, and the analytes were separated on a Dikma Diamonsil C₁₈ (200 mm×4.6 mm i.d., 5 µm) column using a mobile phase comprised of methanol:acetonitrile: 0.5% formic acid (20∶25∶55, v/v/v) at a flow rate of 1 ml/min. Detection was performed on a Finnigan TSQ ripple quadrupole tandem mass spectrometer in negative ion‐selected monitoring mode using electrospray ionization. Good linearity over the range 10–1000 ng/ml for danshensu, salvianolic acid B, and hydroxysafflor yellow A, and 5–500 ng/ml for protocatechuic aldehyde with acceptable accuracy and precision, respectively. All the validation data, such as accuracy, precision, and stability, were within the required limits. It was a potential platform for the pharmacokinetic and absorption, distribution, metabolism, and excretion (ADME) study of multiple constituent traditional Chinese medicine.     

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.     

Development and validation of a RP‐HPLC method for stability‐indicating assay of gemifloxacin mesylate including identification of related substances by LC‐ESI‐MS/MS, 1H and 13C NMR spectroscopy

A validated stability indicating RP‐HPLC assay of gemifloxacin mesylate was developed by separating its related substances on an Inertsil‐ODS3V‐C₁₈ (4.6 × 250 mm; 5 μm) column using 0.1% trifluoroaceticacid (pH 2.5) and methanol as a mobile phase in a gradient elution mode at a flow rate of 1.0 mL/min at 27°C. The column effluents were monitored by a photodiode array detector set at 287 nm. The method was validated in terms of accuracy, precision and linearity as per ICH guidelines. Forced degradation of gemifloxacin (GFX) was carried out under acidic, basic, thermal, photolysis and peroxide conditions and the degradation products were separated and characterized by ESI‐MS/MS, ¹H and ¹³C NMR spectroscopy. The method was successfully applied to the analysis of bulk drugs and the recoveries of gemifloxacin and impurities were in the range of 97.60–102.90 and 96.99–102.10%, respectively. No previous reports were found in the literature on identification of degradation products of gemifloxacin. Copyright © 2011 John Wiley & Sons, Ltd.