Application of LC-NMR and HR-NMR to the characterization of biphenyl impurities in the synthetic route development for vestipitant,a novel NK1 antagonist

Vestipitant (1) is a novel NK1 antagonist currently under investigation for the treatment of CNS disorders and emesis. The first synthetic step comprised a Grignard synthesis. An impurity was identified and initially expected to be a symmetric biphenyl. This paper reports the work to synthesise the supposed structure and the spectroscopic analyses (LC-NMR and HR-NMR) to correctly identify the real structure and understand the chemical pathway of the impurity.     

Identification, isolation and characterization of an unknown impurity of varenicline

An unknown impurity formed during stability sample analysis by a gradient reversed phase ultra-high pressure liquid chromatography (UHPLC) of varenicline tablets at 0.2% level. A simple isocratic preparative method was developed to isolate the unknown impurity with 20 min run time. This unknown impurity was identified and characterized by using spectroscopic techniques. Based on the spectral data, the unknown impurity has been characterized as 4,6,7,8,9,10-hexahydro-1H-6,10-methanopyrazino[2,3-h][3]benzazepine-2,3-dione. The structure of this impurity was also established unambiguously, prepared by isolation and co-injected into UHPLC to confirm the retention time. To the best of our knowledge, this impurity has not been reported elsewhere.     

Pharmaceutical impurity identification: a case study using a multidisciplinary approach

A multidisciplinary team approach to identify pharmaceutical impurities is presented in this article. It includes a representative example of the methodology. The first step is to analyze the sample by LC-MS. If the structure of the unknown impurity cannot be conclusively determined by LC-MS, LC-NMR is employed. If the sample is unsuitable for LC-NMR, the impurity needs to be isolated for conventional NMR characterization. Although the technique of choice for isolation is preparative HPLC, enrichment is often necessary to improve preparative efficiency. One such technique is solid-phase extraction. For complete verification, synthesis may be necessary to compare spectroscopic characteristics to those observed in the original sample. Although not widely practiced, an effective means of getting valuable structural information is to conduct a degradation study on the purified impurity itself. This systematic strategy was successfully applied to the identification of an impurity in the active pharmaceutical ingredient 1-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)-3-[4-(1-hydroxy-1-methyl-ethyl)-furan-2-sulphonylurea. Identification required the use of all of the previously mentioned techniques. The instability of the impurity under acidic chromatographic conditions presented an additional challenge to purification and identification. However, we turned this acidic instability to an advantage, conducting a degradation study of the impurity, which provided extensive and useful information about its structure. The following discussion describes how the information gained from each analytical technique was brought together in a complementary fashion to elucidate a final structure.     

Isolation, synthesis and characterization of impurities in celecoxib, a COX-2 inhibitor

During the impurity profile of Celecoxib, four polar impurities (impurity I, II, III and IV) and one non-polar impurity (impurity V) with respect to Celecoxib were detected by HPLC. LC-MS has been employed in this impurity profile study. The three polar impurities (I, II and III) were found to be process related while impurities (IV and V) turned out to be isomers. The impurities III, IV and V were isolated with the help of preparative HPLC. The structure of impurities III, IV (ortho-isomer) and V (regio-isomer) were confirmed as [5-(4-methylphenyl)-3-trifluoromethyl-1H-pyrazole], 4-[5-(2'-methyl phenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamide, and 4-[4-(4'-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-benzenesulfonamide, respectively. The structures of impurities I, II, III and IV were confirmed by synthesis and structural characterization using spectral data. However, the impurity V was not synthesized.     

Isolation and characterization of benazepril unknown impurity by chromatographic and spectroscopic methods

The presence of unknown impurity of the order of 0.2% was identified in benazepril using liquid chromatographic technique employing binary gradient system comprising acetic acid and ammonia in water and acetonitrile as the mobile phase. LCMS data corresponds to hydroxylated benazepril (OHB) derivative possessing the molecular formula C(24)H(28)N(2)O(6). This impurity was isolated using isocratic system containing ammonium acetate and acetonitrile and the product was characterized using FT-IR, (1)H and (13)C NMR and mass spectroscopy to ascertain the structure of the impurity. The spectroscopic analysis revealed the presence of hydroxyl function on C(17) carbon atom of benazepril molecule. The plausible mechanism for the formation of OHB species is proposed.     

Application of LC-MS/MS for the identification of a polar impurity in mosapride,a gastroprokinetic drug

In the impurity profile of mosapride a polar impurity (0.1%) was detected in HPLC with respect to mosapride. Based on the mass spectral data obtained by LC-MS/MS analysis this impurity structure was characterised as 4-amino-5-chloro-2-ethoxy-N-[[(4-benzyl)-2-morphinyl] methyl] benzamide. It is interesting to note that this impurity is potent analogue of mosapride, which will have much higher gastroprokinetic activity than metoclopramide. This impurity was synthesised from an unambiguous route and confirmed the structure by collecting various spectral data and the formation is discussed. To our knowledge this compound was not reported as process impurity elsewhere.     

Identification and characterization of a principal oxidation impurity in clopidogrel drug substance and drug product

The focus of this study is identification, isolation and characterization of a principal oxidation impurity of clopidogrel which ranged from 0.05 to 0.12% using high performance liquid chromatography. This impurity is considered as principal oxidation impurity as it is observed in oxidative degradation (stress) study. Preparative HPLC with Xterra MS C18 ODB column was used to isolate the impurity. The isolated impurity was co-injected with the sample containing impurities and found the retention time match of the spiked impurities. A thorough study was undertaken to characterize this impurity and based on their spectral data (UV, MS, MSn 1H/13C, DEPT and 2D NMR) the structure was characterized as 5-[1-(2-chlorophenyl)-2-methoxy-2-oxoethyl]-6,7-dihydrothieno[3,2-c]pyridin-5-ium with a molecular weight 320 amu.     

盐酸艾司洛尔注射液中杂质的分离、合成与结构鉴定

目的:研究盐酸艾司洛尔注射液中杂质的结构和产生的原因。方法:采用制备高效液相色谱方法制备分离盐酸艾司洛尔注射液的杂质并采用IR、MS1、H-NMR、13C-NMR等方法进行结构鉴定;合成制备了此杂质并进行结构确证。结果:该杂质为4-{[2-羟基-3-[(1-甲基乙基)氨基]]丙氧基}苯丙酸盐酸盐,为盐酸艾司洛尔注射液在储藏过程的降解产物。结论:本研究可为盐酸艾司洛尔注射液质量评价提供依据。     

Isolation,Identification, and Characterization of an Unknown Impurity in Lovastatin EP

An unknown impurity in the fermentation-based drug substance lovastatin at 0.52 RRT was observed invariably in all batches when analyzed by HPLC as per the PhEur monograph. This impurity was isolated from the impurity-enriched sample using reversed-phase preparative HPLC and characterized by using spectroscopic (PMR, CMR, MASS, and UV) techniques as the structurally-related compound Monacolin-X, having the molecular formula C₂₄H₃₄O₆ and the chemical name 2-methyl-3-oxobutanoic acid 1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-naphthalenyl ester.     

Identification and characterization of potential impurities of quetiapine fumarate

Seven potential impurities, including by-products, starting materials and intermediates were identified in pharmaceutical substance quetiapine fumarate and characterized by spectroscopic methods (MS, IR, NMR). Based on these methods the structures of the impurities were assigned or confirmed as: impurity I: 2-(phenylthio)aniline; impurity II: phenyl N-[2-(phenylthio)phenyl]carbamate; impurity III: N,N'-bis[2-(phenylthio) phenyl]urea; impurity IV: N-[2-(phenylthio)phenyl]-1-piperazinecarboxamide hydrochloride; impurity V: N,N'-bis[(2-phenylthio)phenyl]-1,4-piperazinedicarboxamide; impurity VI: 11-(1-piperazinyl) dibenzo[b,f][1,4]thiazepine fumarate; impurity VII: 1,4-bis(dibenzo[b,f][1,4] thiazepin-11-yl)piperazine. Structural elucidation of compounds, proposed MS fragmentation pathway and possible ways of formation of the impurities are also discussed.     

The structure of tautomycetin, a dialkylmaleic anhydride antibiotic

The chemical structure of tautomycetin (C33H50O10) was determined by chemical degradation and spectroscopic evidence. Tautomycetin exists in methanol-buffer solution (1% diethylamine-formic acid, pH 7.3) as an equilibrium mixture of a 2,3-dialkylmaleic anhydride and its dicarboxylic acid. The structure of tautomycetin is similar to tautomycin in many respects.     

Identification,isolation and characterization of new impurity in rabeprazole sodium

Rabeprazole sodium [1] is a proton pump inhibitor, used as an antiulcerative. During the manufacturing of rabeprazole sodium, we observed an unknown impurity at levels 0.05–0.1% in HPLC analysis along with the known potential impurities. This new unknown impurity was isolated using preparative liquid chromatography. Based on the complete spectral analysis (¹H NMR, ¹³C NMR, DEPT, Mass and IR), this new impurity was designated as 2-[[(3-methyl-4-(methylthio)-2-pyridinyl)methyl]sulfinyl]-1H-benzimidazole (methylthio impurity of rabeprazole). Impurity isolation, structure elucidation and probable formation mechanism was discussed.     

Cytotoxicity, cellular uptake, and DNA interactions of new monodentate ruthenium(II) complexes containing terphenyl arenes

We have compared the cancer cell cytotoxicity, cell uptake, and DNA binding properties of the isomeric terphenyl complexes [(eta(6)-arene)Ru(en)Cl](+), where the arene is ortho- (2), meta- (3), or para-terphenyl (1) (o-, m-, or p-terp). Complex 1, the X-ray crystal structure of which confirms that it has the classical "piano-stool" geometry, has a similar potency to cisplatin but is not cross-resistant and has a much higher activity than 2 or 3. The extent of Ru uptake into A2780 or A2780cis cells does not correlate with potency. Complex 1 binds to DNA rapidly and quantitatively, preferentially to guanine residues, and causes significant DNA unwinding. Circular and linear dichroism, competitive binding experiments with ethidium bromide, DNA melting, and surface-enhanced Raman spectroscopic data are consistent with combined intercalative and monofunctional (coordination) binding mode of complex 1. This unusual DNA binding mode may therefore make a major contribution to the high potency of complex 1.     

Barbatoside, a new Iridoid glucoside from Penstemon barbatus

From leaves of Penstemon barbatus Nutt. a new iridoid glucoside has been isolated. The structure was established mainly by spectroscopic methods ( (1)H-NMR, (13)C-NMR, IR, MS).     

Seitomycin: isolation,structure elucidation and biological activity of a new angucycline antibiotic from a terrestrial Streptomycete

A new antibiotic, named seitomycin (1c), and the known microbial metabolite tetrangulol methyl ether (2) were found in the ethyl acetate extract of two terrestrial Streptomyces sp. isolates. The structure of the new antibiotic was elucidated by spectroscopic studies and by comparison of the NMR data with the structurally related hatomarubigin C (1a) and SM-196 B (1b). Seitomycin (1c) showed moderate antimicrobial and weak phytotoxic activity, similar to tetrangulol methyl ether (2).     

Barbatoside, a New Iridoid Glucoside from Penstemon barbatus.]

From leaves of PENSTEMON BARBATUS N UTT. a new iridoid glucoside has been isolated. The structure was established mainly by spectroscopic methods ( (1)H-NMR, (13)C-NMR, IR, MS).     

Identification, isolation and characterization of a new degradation product in sultamicillin drug substance

A new degradant of sultamicillin drug substance was found during the gradient reverse phase HPLC analysis of stability storage samples. The level of this degradant impurity was observed up to 1.0%. The impurity (formaldehyde adduct with 5-oxo-4-phenylimidazolidin-1-yl moiety) was identified by LC/MS and was characterized by ((1)H NMR, (13)C NMR, 2D-NMR ((1)H-(1)H COSY, NOESY, HSQC and HMBC), LC/MS/MS, MS/TOF, elemental analysis and IR. This impurity was prepared by isolation and co-injected into HPLC system to confirm the retention time.     

Identification, isolation and characterization of potential degradation product in risperidone tablets

One unknown impurity (degradation product) present at a level below 0.1% in the initial samples increased to a level of 0.5% in 6M/40 degrees C/75% RH stability samples of risperidone tablets was detected by gradient reverse-phase high-performance liquid chromatography (HPLC). This impurity was isolated using reverse-phase preparative liquid chromatography. Based on the spectral data the structure of this impurity is characterized as 3-[2-[4-[6-fluoro-1,3-benzoxazol-2-yl]piperidin-1-yl]ethyl]-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a] pyrimidin-4-one. Structural elucidation of this impurity by spectral data ((1)H NMR, (13)C NMR, DEPT, MS and IR), formation and mechanism has been discussed in detail.     

Identification,isolation, characterization and response factor determination of process-related impurity in meprobamate drug substance

This paper describes identification and characterization of a process-related impurity of meprobamate drug substance observed in HPLC-UV method. Forced degradation studies were carried out under acidic, basic, oxidation, light and thermal conditions to assess the nature of the impurity. The pure impurity was obtained by preparative LC isolation and analyzed by NMR and mass. Structural elucidation by spectral data and formation of this impurity were discussed in detail. The structure of the process-related impurity was established as carbamic acid-2-carbamoyloxymethyl-2-methyl-pent-3-enyl ester (olefin). Also, the relative response factor, linearity, detection limit (DL), quantitation limit (QL) and recovery were determined for meprobamate and the impurity. Good linearity was obtained for the impurity over the concentration range of 0.03–0.20% (w/w) with the coefficient of determination (r²) of 0.999. The DL and QL of olefin impurity were 0.0003 and 0.001% (w/w), respectively. The isolated impurity was co-injected with meprobamate sample to confirm the retention time in HPLC.     

Trans-dimer D, a novel dimeric sesquiterpene with a bis-bisabolene skeleton from a Hainan sponge Axinyssa variabilis

A novel bisabolene sesquiterpene dimer named trans-dimer D (1) and its diastereoisomer trans-dimer C (2) reported in our previous work have been isolated as an inseparable mixture in a ratio of 1.5:1 from the South China Sea sponge Axinyssa variabilis. The structure of 1 was determined on the basis of extensive spectroscopic analysis and by comparison of its NMR spectral data with those of structurally related compounds. Compound 1 represents the fourth example of a sesquiterpene dimer with a bis-bisabolene skeleton.