Studies on the metabolism and toxicological detection of glaucine,an isoquinoline alkaloid from Glaucium flavum (Papaveraceae), in rat urine using GC‐MS,LC‐MSn and LC‐high‐resolution MSn

Glaucine ((S)‐5,6,6a,7‐tetrahydro‐1,2,9,10‐tetramethoxy‐6‐methyl‐4H‐dibenzo [de,g]quinoline) is an isoquinoline alkaloid and main component of Glaucium flavum (Papaveraceae). It was described to be consumed as recreational drug alone or in combination with other drugs. Besides this, glaucine is used as therapeutic drug in Bulgaria and other countries as cough suppressant. Currently, there are no data available concerning metabolism and toxicological analysis of glaucine. To study both, glaucine was orally administered to Wistar rats and urine was collected. For metabolism studies, work‐up of urine samples consisted of protein precipitation or enzymatic cleavage followed by solid‐phase extraction. Samples were afterwards measured by liquid chromatography (LC) coupled to low or high‐resolution mass spectrometry (HR‐MS). The phase I and II metabolites were identified by detailed interpretation of the corresponding fragmentations, which were further confirmed by determination of their elemental composition using HR‐MS. From these data, the following metabolic pathways could be proposed: O‐demethylation at position 2, 9 and 10, N‐demethylation, hydroxylation, N‐oxidation and combinations of them as well as glucuronidation and/or sulfation of the phenolic metabolites. For monitoring a glaucine intake in case of abuse or poisoning, the O‐ and N‐demethylated metabolites were the main targets for the gas chromatography‐MS and LC‐MSⁿ screening approaches described by the authors. Both allowed confirming an intake of glaucine in rat urine after a dose of 2 mg/kg body mass corresponding to a common abuser's dose. Copyright © 2013 John Wiley & Sons, Ltd.     

新型除草剂及其杂质的LC-MS和GC-MS分析

High performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) have been utilized to analyze the synthesized 2-(2-arylaminomethylphenoxy)pyrimidine derivatives, which are a new kind of environmentally benign herbicides and have passed the temporary pesticide registration. The identification of main product and impurities has been achieved according to the UV and mass spectra. Moreover, one impurity, introduced by the raw material in the last step of the synthetic route, was identified by GC-MS analysis. It can be concluded that the combination of chromatography and mass spectrometry, including LC-MS and GC-MS, provided a vital tool of the pesticide science.     

MSn,LC‐MS‐TOF and LC‐PDA studies for identification of new degradation impurities of bupropion

Three new degradation impurities of bupropion were characterized through high performance liquid chromatography coupled to photodiode array detection and to time‐of‐flight mass spectrometry. Bupropion was subjected to the ICH prescribed stress conditions. It degraded to seven impurities (I–VII) in alkaline hydrolytic conditions which were optimally resolved on an XTerra C₁₈ column (250 × 4.6 mm, 5 µm) with a ternary mobile phase comprising ammonium formate (20 mm , pH 4.0), methanol and acetonitrile (75:10:15, v/v). The degradation impurities (III–V and VII) were characterized on the basis of mass fragmentation pattern of drug, accurate mass spectral and photodiode array data of the drug and degradation impurities. Compound V was found to be a known degradation impurity [1‐hydroxy‐1‐(3‐chlorophenyl)propan‐2‐one], whereas III, IV and VII were characterized as 2‐hydroxy‐2‐(3′‐chlorophenyl)‐3,5,5‐trimethylmorpholine, (2,4,4‐trimethyl‐1,3‐oxazolidin‐2‐yl)(3‐chlorophenyl)‐methanone and 2‐(3′‐chlorophenyl)‐3,5,5‐trimethylmorphol‐2‐ene, respectively. Compound III was a known metabolite of the drug. This additional information on the degradation impurities can help in the development of a new stability‐indicating assay method to monitor the stability of the drug product during its shelf‐life as well as in development of a drug product with increased shelf‐life. Copyright © 2013 John Wiley & Sons, Ltd.     

GC‐MS of amaryllidaceous galanthamine‐type alkaloids

Galanthamine‐type alkaloids produced by plants of the Amaryllidaceae family are potent acetylcholinesterase inhibitors. One of them, galanthamine, has been marketed as a hydrobromide salt for the treatment of Alzheimer's disease. In the present work, gas chromatography with electron impact mass spectrometry (GC‐EIMS) fragmentation of 12 reference compounds isolated from various amaryllidaceous plants and identified by spectroscopic methods (1D and 2D nuclear magnetic resonance, circular dichroism, high‐resolution MS (HRMS) and EIMS) was studied by tandem mass spectrometry (GC‐MS/MS) and accurate mass measurements (GC‐HRMS). The studied compounds showed good peak shape and efficient GC separation with a GC‐MS fragmentation pattern similar to that obtained by direct insertion probe. With the exception of galanthamine‐N‐oxide and N‐formylnorgalanthamine, the galanthamine‐type compounds showed abundant [M]^+. and [M‐H]⁺ ions. A typical fragmentation pattern was also observed, depending on the substituents of the skeleton. Based on the fragmentation pathways of reference compounds, three other galanthamine‐type alkaloids, including 3‐O‐(2′‐butenoyl)sanguinine, which possesses a previously unelucidated structure, were identified in Leucojum aestivum ssp. pulchelum, a species endemic to the Balearic islands. GC‐MS can be successfully applied to Amaryllidaceae plant samples in the routine screening for potentially new or known bioactive molecules, chemotaxonomy, biodiversity and identification of impurities in pharmaceutical substances. Copyright © 2012 John Wiley & Sons, Ltd.     

Studies on the metabolism of the Δ9‐tetrahydrocannabinol precursor Δ9‐tetrahydrocannabinolic acid A (Δ9‐THCA‐A) in rat using LC‐MS/MS,LC‐QTOF MS and GC‐MS techniques

In Cannabis sativa, Δ9‐Tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A) is the non‐psychoactive precursor of Δ9‐tetrahydrocannabinol (Δ9‐THC). In fresh plant material, about 90% of the total Δ9‐THC is available as Δ9‐THCA‐A. When heated (smoked or baked), Δ9‐THCA‐A is only partially converted to Δ9‐THC and therefore, Δ9‐THCA‐A can be detected in serum and urine of cannabis consumers. The aim of the presented study was to identify the metabolites of Δ9‐THCA‐A and to examine particularly whether oral intake of Δ9‐THCA‐A leads to in vivo formation of Δ9‐THC in a rat model. After oral application of pure Δ9‐THCA‐A to rats (15 mg/kg body mass), urine samples were collected and metabolites were isolated and identified by liquid chromatography‐mass spectrometry (LC‐MS), liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and high resolution LC‐MS using time of flight‐mass spectrometry (TOF‐MS) for accurate mass measurement. For detection of Δ9‐THC and its metabolites, urine extracts were analyzed by gas chromatography‐mass spectrometry (GC‐MS). The identified metabolites show that Δ9‐THCA‐A undergoes a hydroxylation in position 11 to 11‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A (11‐OH‐Δ9‐THCA‐A), which is further oxidized via the intermediate aldehyde 11‐oxo‐Δ9‐THCA‐A to 11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A‐COOH). Glucuronides of the parent compound and both main metabolites were identified in the rat urine as well. Furthermore, Δ9‐THCA‐A undergoes hydroxylation in position 8 to 8‐alpha‐ and 8‐beta‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A, respectively, (8α‐Hydroxy‐Δ9‐THCA‐A and 8β‐Hydroxy‐Δ9‐THCA‐A, respectively) followed by dehydration. Both monohydroxylated metabolites were further oxidized to their bishydroxylated forms. Several glucuronidation conjugates of these metabolites were identified. In vivo conversion of Δ9‐THCA‐A to Δ9‐THC was not observed. Copyright © 2009 John Wiley & Sons, Ltd.     

Validated LC‐MS/MS assay for the quantitative determination of vardenafil in human plasma and its application to a pharmacokinetic study

A sensitive high‐performance liquid chromatography–tandem mass spectrometric (HPLC‐MS/MS) assay has been developed for the quantitative analysis of vardenafil in human plasma. Vardenafil and the internal standard, alprazolam, were extracted from 0.2 mL aliquots of alkalinized plasma by a single solvent extraction into hexane : dichloromethane. Reversed‐phase chromatographic separation was affected by gradient elution with mobile phases consisting of 10 mM ammonium formate pH 7.0 (solvent A) and methanol (100%, solvent B), delivered at a flow rate of 0.4 mL/min. The analytes were detected by using an electrospray ion source on a 4000 QTrap triple quadrupole mass spectrometer operating in positive ionization mode. The mass transitions were m/z 489.3 → 312.2 for vardenafil and m/z 309.2 → 281.0 for alprazolam. The assay was linear over the concentration range of 0.2–100 ng/mL, with correlation coefficients ≥0.995. The intra‐ and inter‐day precision was less than 5.4% in terms of relative standard deviation and the accuracy was within 12.7% in terms of relative error. The lower limit of quantitation was set at 0.2 ng/mL. The high sensitivity and acceptable performance of the assay allowed its application to the analysis of plasma samples obtained following the oral administration of vardenafil to healthy male volunteers in a pharmacokinetic study. Copyright © 2009 John Wiley & Sons, Ltd.     

Metabolic profile of glyburide in human liver microsomes using LC‐DAD‐Q‐TRAP‐MS/MS

The sulfonylurea urea drug glyburide (glibenclamide) is widely used for the treatment of diabetes milletus and gestational diabetes. In previous studies monohydroxylated metabolites were identified and characterized for glyburide in different species, but the metabolite owing to the loss of cyclohexyl ring was identified only in mouse. Glyburide upon incubation with hepatic microsomes resulted in 10 metabolites for human. The current study identifies new metabolites of glyburide along with the hydroxylated metabolites that were reported earlier. The newly identified drug metabolites are dihydroxylated metabolites, a metabolite owing to the loss of cyclohexyl ring and one owing to hydroxylation with dehydrogenation. Among the 10 identified metabolites, there were six monohydroxylated metabolites, one dihydroxylated metabolite, two metabolites owing to hydroxylation and dehydrogenation, and one metabolite owing to the loss of cyclohexyl ring. New metabolites of glyburide were identified and characterized using liquid chromatography–diode array detector–quadruple‐ion trap–mass spectrometry/mass spectrometry (LC‐DAD‐Q‐TRAP‐MS/MS). An enhanced mass scan–enhanced product ion scan with information‐dependent acquisition mode in a Q‐TRAP‐MS/MS system was used to characterize the metabolites. Liquid chromatography with diode array detection was used as a complimentary technique to confirm and identify the metabolites. Metabolites formed in higher amounts were detected in both diode array detection and mass spectrometry detection. Copyright © 2012 John Wiley & Sons, Ltd.     

Metabolite analysis in Curcuma domestica using various GC‐MS and LC‐MS separation and detection techniques

The metabolic profile of polar (methanol) and non‐polar (hexane) extracts of Curcuma domestica, a widely used medicinal plant, was established using various different analytical techniques, including GC‐FID, GC‐MS, HR‐GC‐MS and analytical HPLC‐ESI‐MS/MS by means of LTQ‐Orbitrap technology. The major non‐volatile curcuminoids curcumin, demethoxycurcumin and bisdemethoxycurcumin were identified when their chromatographic and precursor ion masses were compared with those of authentic standard compounds. In this paper we describe for the first time a GC/MS‐based method for metabolic profiling of the hydrophilic extract. We also identified 61 polar metabolites as TMS derivatives. Copyright © 2009 John Wiley & Sons, Ltd.     

Combined quantification of paclitaxel,docetaxel and ritonavir in human feces and urine using LC‐MS/MS

A combined assay for the determination of paclitaxel, docetaxel and ritonavir in human feces and urine is described. The drugs were extracted from 200 μL urine or 50 mg feces followed by high‐performance liquid chromatography analysis coupled with positive ionization electrospray tandem mass spectrometry. The validation program included calibration model, accuracy and precision, carry‐over, dilution test, specificity and selectivity, matrix effect, recovery and stability. Acceptance criteria were according to US Food and Drug Administration guidelines on bioanalytical method validation. The validated range was 0.5–500 ng/mL for paclitaxel and docetaxel, 2–2000 ng/mL for ritonavir in urine, 2–2000 ng/mg for paclitaxel and docetaxel, and 8–8000 ng/mg for ritonavir in feces. Inter‐assay accuracy and precision were tested for all analytes at four concentration levels and were within 8.5% and <10.2%, respectively, in both matrices. Recovery at three concentration levels was between 77 and 94% in feces samples and between 69 and 85% in urine samples. Method development, including feces homogenization and spiking blank urine samples, are discussed. We demonstrated that each of the applied drugs could be quantified successfully in urine and feces using the described assay. The method was successfully applied for quantification of the analytes in feces and urine samples of patients. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitation of melatonin and n‐acetylserotonin in human plasma by nanoflow LC‐MS/MS and electrospray LC‐MS/MS

Melatonin (MEL) and its chemical precursor N‐acetylserotonin (NAS) are believed to be potential biomarkers for sleep‐related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC‐MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies, and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays, plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1 × 100 mm, 3.5 µm) or on a polyimide‐coated, fused‐silica capillary self‐packed with 17 cm AquaC18 (3 µm, 125 Å). Quantitation was done using the SRM transitions m/z 233 → 174 and m/z 219 → 160 for MEL and NAS, respectively. The analytical response ratio versus concentration curves were linear for MEL (nanoflow LC: 11.7–1165 pg/mL, LC: 1165–116500 pg/mL) and for NAS (nanoflow LC: 11.0–1095 pg/mL). Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of a sensitive assay for the quantification of imatinib using LC/LC‐MS/MS in human whole blood and cell culture

We developed and validated a semi‐automated LC/LC‐MS/MS assay for the quantification of imatinib in human whole blood and leukemia cells. After protein precipitation, samples were injected into the HPLC system and trapped onto the enrichment column (flow 5 mL/min); extracts were back‐flushed onto the analytical column. Ion transitions [M + H]⁺ of imatinib (m/z = 494.3 → 394.3) and its internal standard trazodone (372.5 → 176.3) were monitored. The range of reliable response was 0.03–75 ng/mL. The inter‐day precisions were: 8.4% (0.03 ng/mL), 7.2% (0.1 ng/mL), 6.5% (1 ng/mL), 8.2% (10 ng/mL) and 4.3% (75 ng/mL) with no interference from ion suppression. Autosampler stability was 24 hs and samples were stable over three freeze–thaw cycles. This semi‐automated method is simple with only one manual step, uses a commercially available internal standard, and has proven to be robust in larger studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparison of sulfo‐conjugated and gluco‐conjugated urinary metabolites for detection of methenolone misuse in doping control by LC‐HRMS,GC‐MS and GC‐HRMS

Methenolone (17β‐hydroxy‐1‐methyl‐5α‐androst‐1‐en‐3‐one) misuse in doping control is commonly detected by monitoring the parent molecule and its metabolite (1‐methylene‐5α‐androstan‐3α‐ol‐17‐one) excreted conjugated with glucuronic acid using gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography mass spectrometry (LC‐MS) for the parent molecule, after hydrolysis with β‐glucuronidase. The aim of the present study was the evaluation of the sulfate fraction of methenolone metabolism by LC‐high resolution (HR)MS and the estimation of the long‐term detectability of its sulfate metabolites analyzed by liquid chromatography tandem mass spectrometry (LC‐HRMSMS) compared with the current practice for the detection of methenolone misuse used by the anti‐doping laboratories. Methenolone was administered to two healthy male volunteers, and urine samples were collected up to 12 and 26 days, respectively. Ethyl acetate extraction at weak alkaline pH was performed and then the sulfate conjugates were analyzed by LC‐HRMS using electrospray ionization in negative mode searching for [M‐H]^? ions corresponding to potential sulfate structures (comprising structure alterations such as hydroxylations, oxidations, reductions and combinations of them). Eight sulfate metabolites were finally detected, but four of them were considered important as the most abundant and long term detectable. LC clean up followed by solvolysis and GC/MS analysis of trimethylsilylated (TMS) derivatives reveal that the sulfate analogs of methenolone as well as of 1‐methylene‐5α‐androstan‐3α‐ol‐17‐one, 3z‐hydroxy‐1β‐methyl‐5α‐androstan‐17‐one and 16β‐hydroxy‐1‐methyl‐5α‐androst‐1‐ene‐3,17‐dione were the major metabolites in the sulfate fraction. The results of the present study also document for the first time the methenolone sulfate as well as the 3z‐hydroxy‐1β‐methyl‐5α‐androstan‐17‐one sulfate as metabolites of methenolone in human urine. The time window for the detectability of methenolone sulfate metabolites by LC‐HRMS is comparable with that of their hydrolyzed glucuronide analogs analyzed by GC‐MS. The results of the study demonstrate the importance of sulfation as a phase II metabolic pathway for methenolone metabolism, proposing four metabolites as significant components of the sulfate fraction. Copyright © 2015 John Wiley & Sons, Ltd.     

Structural motifs in primary oxidation products of palmitoyl‐arachidonoyl‐phosphatidylcholines by LC‐MS/MS

Oxidative modifications to phospholipids (OxPL) play a major role in modulating signaling events in inflammation and infection, and complete understanding on the induced biological effects can only be understood based on knowledge of the oxidative motifs present. Specific neutral losses observed in tandem mass spectrometry data (LC‐MS/MS) of primary peroxidation products in oxidized palmitoyl‐arachidonoyl‐phosphatidylcholines (OxPAPC) provide information on the prevailing structural motifs regarding the oxidized acyl carbon chain, the nature of oxidized group and the site of carbon oxidation. The higher hydrophobicity of hydroperoxides compared to di‐hydroxy derivatives under reverse‐phase conditions together with specific fragmentation patterns enabled the identification of 12 structurally different OxPAPC structural (di‐hydroxy and hydroperoxide derivatives) and positional isomers as well as the presence of poly‐hydroxy together with isoprostanes derivatives. The fragmentation patterns described in quadrupole time‐of‐flight and linear ion trap instruments complement the m/z value and retention time parameters in the identification of oxidative composition in OxPAPC products becoming a valuable tool for the exploratory screening of oxidized phosphatidylcholines in OxPAPC extracts, distinction of native and modified PC isobaric structures in complex samples contributing to the increased understanding of redox lipidomics in inflammation and infection. Copyright © 2013 John Wiley & Sons, Ltd.     

Separation and identification of enzymatically prepared dephosphorylated products of myo‐inositolhexakisphosphate using LC‐MS

LC‐MS technique described here is a new way for the separation and direct determination of UV–Vis insensitive inositol phosphates (InsP₂‐InsP₆). This circumvents the need of radioisotopic labeling and post‐column derivatization techniques. The method involves separation of various enzymatically dephosphorylated derivatives of InsP₆ on C₁₈‐column using MeOH/H₂O (30:70 v/v) and their identification using electron spray ionization MS in positive ion mode (+pESI‐MS). The LC‐MS studies revealed that the purified phytase from Aspergillus niger van Teighem hydrolyzes InsP₆ in a sequential manner leading to InsP₂ (InsP₂·2Na, t_R 4.4–4.54 min, base peak m/z 382.9) as the end product.     

Determination of penicillins in milk using LC‐UV,LC‐MS and LC‐MS/MS

The aim of this work is to establish a method for the simultaneous determination of eight penicillins in milk samples by LC‐UV, LC‐MS and LC‐MS/MS. The procedure involves a step for clean‐up and to preconcentrate the analytes by SPE and a subsequent chromatographic analysis. LC‐UV, LC‐MS and LC‐MS/MS have been used for the simultaneous quantification of penicillins in milk. The proposed methods have been validated according to the EU guideline and present LOQ below the maximum limits of residues (MRLs) established by the European Union for penicillins in milk. The developed methods were applied to different milk samples obtained from cows medicated with penicillins.     

Determination of lumiracoxib by a validated stability‐indicating MEKC method and identification of its degradation products by LC‐ESI‐MS studies

A stability‐indicating MEKC method was developed and validated for the analysis of lumiracoxib (LMC) in pharmaceutical formulations using nimesulide as the internal standard (IS). Optimal conditions for the separation of LMC and degradation products were investigated. The method employed 50 mM borate buffer and 50 mM anionic detergent SDS solution at pH 9.0. MEKC method was performed on a fused‐silica capillary (50 μm id; effective length, 40 cm) maintained at 30°C. The applied voltage was 20 kV and photodiode array (PDA) detector was set at 208 nm. The method was validated in accordance with the International Conference on Harmonisation requirements. The stability‐indicating capability of the method was established by enforced degradation studies combined with peak purity assessment using PDA detection. The degradation products formed under stressed conditions were investigated by LC‐ESI‐MS and the two degraded products were identified. MEKC method was linear over the concentration range of 5–150 μg/mL (r²=0.9999) of LMC. The method was precise, accurate, with LOD and LOQ of 1.34 and 4.48 μg/mL, respectively. The robustness was proved by a fractional factorial design evaluation. The proposed MEKC method was successfully applied for the quantitative analysis of LMC in tablets to support the quality control.     

LC‐PDA‐ESI‐MSn analysis of phenolic and iridoid compounds from Globularia spp.

Plants produce a great number of metabolites with potentially useful biological activities. Species from the genus Globularia (Plantaginaceae) are known as sources of different phenolic and iridoid compounds. Globularia alypum L. is a medicinal plant used as a healing agent in many Mediterranean countries. Similarities in phytochemical composition are often observed for related species. For Globularia spp., such findings were mostly based on identification of several isolated compounds from distinct species. To our knowledge, this is the first study that enables simultaneous comparison of phytochemical profiles from several members of the genus Globularia. Liquid chromatography‐photodiode array detection‐electrospray ionization‐tandem mass spectrometry was used for the analysis of methanolic extracts of aerial parts obtained from four Globularia species (G. alypum, G. punctata, G. cordifolia and G. meridionalis). In total, 85 compounds were identified or tentatively identified based on comparison of their retention time, UV and MSⁿ (up to MS⁴) spectra to those of standard compounds and/or to literature data. Among these, high relative amounts of bioactive molecules such as globularin, globularifolin, asperuloside and verbascoside (acteoside) were found. Apart from providing new insights into the phytochemistry and chemotaxonomy of selected Globularia species, results of this study complement existing MS/MS spectral data and could enable easier mass spectrometric profiling of certain bioactive compounds such as iridoids and phenylethanoids in related plant species, genera and families. Copyright © 2016 John Wiley & Sons, Ltd.     

Positional isomer differentiation of synthetic cannabinoid JWH‐081 by GC‐MS/MS

Like many new designer drugs of abuse, synthetic cannabinoids (SC) have structural or positional isomers which may or may not all be regulated under law. Differences in acute toxicity may exist between isomers which impose further burden in the fields of forensic toxicology, medicine and legislation. Isomer differentiation therefore becomes crucial from these standpoints as new designer drugs continuously emerge with just minor positional modifications to their preexisting analogs. The aim of this study was to differentiate the positional isomers of JWH‐081. Purchased standard compounds of JWH‐081 and its positional isomers were analyzed by gas chromatography‐electron ionization‐mass spectrometry (GC‐EI‐MS) first in scan mode to investigate those isomers who could be differentiated by EI scan spectra. Isomers with identical or near‐identical EI spectra were further subjected to GC‐tandem mass spectrometry (MS/MS) analysis with appropriate precursor ions. EI scan was able to distinguish 3 of the 7 isomers: 2‐methoxy, 7‐methoxy and 8‐methoxy. The remaining isomers exhibited near‐identical spectra; hence, MS/MS was performed by selecting m/z 185 and 157 as precursor ions. 3‐Methoxy and 5‐methoxy isomers produced characteristic product ions that enabled the differentiation between them. Product ion spectrum of 6‐methoxy isomer resembled that of JWH‐081; however, the relative ion intensities were clearly different from one another. The combination of EI scan and MS/MS allowed for the regioisomeric differentiation of the targeted compounds in this study. Copyright © 2015 John Wiley & Sons, Ltd.     

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.