A validated, stability-indicating HPLC method for the determination of dexamethasone related substances on dexamethasone-coated drug-eluting stents

An HPLC method was developed and validated to determine trace amounts of dexamethasone related substances on dexamethasone-coated drug-eluting stents. Separation of dexamethasone from its major process impurities and degradation products was achieved on a Zorbax Eclipse XDB C8 column using gradient elution and UV detection at 239 nm. The method was validated according to ICH guideline requirements. In addition, stent extraction efficiency, solution stability and method robustness were evaluated. The method was determined to be linear in the range of 0.01-0.30 microg ml(-1) for the quantitation of major dexamethasone related substances. Method accuracy was assessed by spiking dexamethasone acetate at three levels over the range of 0.025-0.175 microg ml(-1). The dexamethasone acetate recovery ranged from 89.6 to 105.8%. The intermediate precision over the three levels was less than 6% (n=9). The method was also shown to be repeatable at concentration levels of 0.025, 0.125 and 0.175 microg ml(-1) dexamethasone with relative standard deviation values of 4.1, 1.7 and 1.6%, respectively. The method was found to be specific, stability-indicating, and sensitive with a detection limit of 0.008 microg ml(-1) and a quantitation limit of 0.025 microg ml(-1) dexamethasone. Finally, the method was demonstrated to be robust, resistant to small variations of chromatographic variables such as pH, mobile phase organic/aqueous composition and column temperature. Identifying unknown dexamethasone degradation products in dexamethasone-coated drug-eluting stents was of critical interest to ensure product quality, since degradants have a significant impact on safety, efficacy, and product storage and handling. The developed chromatographic method was designed to be compatible with mass spectrometric detection. This paper also discusses using this chromatographic method coupled to an ion-trap LCQ mass spectrometer to elucidate proposed structures for four major dexamethasone degradants.     

雷帕霉素药物洗脱支架在STEMI合并糖尿病患者急诊PCI中的应用

目的 探讨雷帕霉素药物洗脱支架在急性ST段抬高型心肌梗死（STEMI）合并糖尿病患者急诊经皮冠状动脉介入（PCI）中应用的安全性与有效性.方法 选取本院2010年7月～2013年7月收治的STEMI合并2型糖尿病行雷帕霉素药物洗脱支架置入术患者（79例）作为试验组,选取同期收治的STEMI合并2型糖尿病行金属裸支架置入术患者（43例）作为对照组,对比两组患者术后12个月不良心血管事件发生率及术后即刻、24 h、48 h、72 h血小板聚集情况.结果 试验组患者术后12个月死亡、再梗死、再次靶血管重建等主要不良心血管事件发生率均明显低于对照组,差异有统计学意义（P＜0.05）.两组患者术前即刻、术后即刻及术后24 h血小板聚集情况差异无统计学意义（P＞0.05）,试验组患者术后48、72 h血小板聚集情况明显优于对照组,差异有统计学意义（P＜0.05）.两组患者术后24 h、术后1个月等近期再狭窄发生率差异无统计学意义（P＞0.05）,试验组患者术后6、12个月再狭窄发生率（8.86％、11.39％）明显低于对照组（25.58％、32.56％）,差异有统计学意义（P＜0.05）.结论 STEMI合并糖尿病患者行急诊PCI术后置入支架能在一定程度上改善心肌再灌注情况,且雷帕霉素药物洗脱支架相较于金属裸支架具有更好的远期疗效,其远期不良心血管事件发生率明显偏低,心肌组织水平再灌注情况优势明显,有助于改善患者术后的生活水平.     

Identification and structural elucidation of an unknown impurity in carbamazepine active pharmaceutical ingredient by liquid chromatography-tandem mass spectrometry and semi-preparative chromatographic isolation

Two impurities were detected in the HPLC analysis of crude carbamazepine active pharmaceutical ingredient. One of the impurities of the order of 0.5% was found to be unknown and has not been reported previously. A LC-MS compatible reverse phase isocratic method was developed and tandem mass spectrometry was performed using electrospray ionization source and ion trap mass analyzer. Isolation of unknown impurity was performed by semi-preparative HPLC followed by characterization using nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (FT-IR) and elemental analysis (CHNS) confirmed its structure as tetrabenzo[b,f,b′f′]azepino[4′,5′:4,5]thieno[2,3-d]azepine-3,9-dicarboxamide. A plausible mechanism for the formation of this impurity is proposed.     

Structural elucidation of a process-related impurity in ezetimibe by LC/MS/MS and NMR

A major process-related impurity associated with the synthesis of ezetimibe was detected by LC–MS. The isolated impurity was found not to have been previously reported. Based on LC/MS/MS studies and accurate mass data, the structure of that impurity was proposed to be 2-(4-hydroxybenzyl)-N,5-bis-(4-fluorophenyl)-5-hydroxypentanamide. The postulated structure was unambiguously confirmed with the help of the NMR and IR analyses of a synthetically obtained sample. The chemical shift of the labile proton of that new entity was assigned by a 2D-NOESY NMR experiment. A rationalization for the formation of this impurity is provided.     

双氢青蒿素差向异构体转化的研究

目的:对双氢青蒿素β型差向异构体向α型转化过程进行分析。方法:建立高效液相色谱法分离异构体。色谱柱:Kromasil C_(18)柱(250mm×4.6mm,5μm),流动相:乙腈-水(80:20),流速:1.0mL·min~(-1),进样量:20μL,检测波长:216nm。采用LC/MS,LC/NMR对其异构体进行了确证。LC/MS:ESI,扫描:50～1000m/z,干燥气:N_2(10L·min~(-1)),温度:350℃,电压:4000V。LC/NMR:500MHz,溶剂:甲醇。结果:双氢青蒿素在有机溶剂中由β型差向异构体向α型转化并且在一定条件下达到平衡。结论:将双氢青蒿素的α型和β型差向异构体的转化条件及其历程作为双氢青蒿素的手性分离。     

Detection, isolation and characterization of principal synthetic route indicative impurities in verapamil hydrochloride

Two unknown impurities were detected in verapamil hydrochloride bulk drug using isocratic reversed-phase high performance liquid chromatography (HPLC). These impurities were isolated by preparative HPLC. Spectral data for the isolated impurities were collected. Based on the spectral data derived from two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy and mass spectrometry (MS), impurity-1 and impurity-2 were characterized as 2-(3,4-dimethoxyphenyl)-3-methylbut-2-enenitrile and 2-(3,4-dimethoxyphenyl)-2-isopropyl-3-methylbutanenitrile, respectively.     

流通池法测试雷帕霉素支架涂药工艺稳定性初探

目的:用美国药典流通池方法测试雷帕霉素药物洗脱冠脉支架体外释放度,作为企业出厂检验方法,以评价其工艺稳定性。方法:用美国药典释放度测定第4法规定的溶出装置联机紫外分光光度计。释放介质为2%十二烷基硫酸钠溶解在10%乙腈-PBS(pH=5.0)的缓冲液;检测波长为280 nm。结果:相同批号(n=6)和不同批号(n=3)的药物支架的雷帕霉素在24 h的释放曲线一致。结论:本法适合评价冠脉支架涂药的工艺稳定性,快速简便,符合美国药典流通池法的要求,检测结果较为满意。     

Synthesis and structural identification of fluorine‐18 labeled parathyroid hormone

Parathyroid hormone (PTH) is an 84 amino acid peptide hormone that plays a key role in bone and mineral metabolism. The biological actions of PTH are mediated via the N‐terminal PTH(1–34) fragment, serving as the PTH receptor‐binding sequence, and which is therefore used clinically to treat conditions of low bone mass such as osteoporosis. In this study, PTH(1–34) was conjugated with non‐radioactive (stable F isotope) N‐succinimidyl 4‐fluorobenzoate (SFB) leading to three isomeric mono‐fluorobenzoated (FBz) PTH followed by Liquid chromatography‐Tandem mass spectrometry (LC‐MS/MS) assisted structural identification. Corresponding [¹⁸F]SFB‐labeled PTH derivatives were prepared respectively and the Lys¹³ site‐specific labeled [¹⁸F]FBz PTH was isolated by HPLC with radiochemical purity >99% and specific activity of 2.78 GBq/µmol, suitable for future application with in vivo pharmacokinetic/pharmacodynamic studies of PTH, using preclinical Positron Emission Tomography Computed Tomography (PET/CT) imaging.     

Isolation and characterization of a potential process related impurity of phenazopyridine HCl by preparative HPLC followed by MS–MS and 2D-NMR spectroscopy

During the process development of phenazopyridine HCl bulk drug, a potential impurity was detected in the routine impurity profiles by HPLC. Using MS–MS and multidimensional NMR techniques, the trace level impurity was unambiguously identified to be 3-phenyl-5-phenylazo-pyridine-2,6-diamine after its isolation from phenazopyridine HCl by semi-preparative HPLC. The formation of the impurity was discussed. To our knowledge, it is a novel impurity not reported elsewhere.     

Strategy for identification and characterization of small quantities of drug degradation products using LC and LC-MS: application to valsartan, a model drug

The present study demonstrates the applicability of a strategy involving use of liquid chromatography (LC) and liquid chromatography-mass spectrometry (LC-MS) techniques for the identification and characterization of minute quantities of degradation products, without their isolation from the reaction matrix in pure form. Valsartan was used as a model drug. It was subjected to forced degradation studies under the International Conference on Harmonisation (ICH) prescribed conditions of hydrolysis (acid, base and neutral), photolysis, oxidation and thermal stress. The drug showed lability under acid/neutral hydrolytic and photolytic conditions, while it was stable to base hydrolytic, oxidative and thermal stress. Three small degradation products were formed, which were separated on a C-18 column using a gradient method. The same were characterized with the help of their fragmentation pattern and accurate masses obtained upon LC-MS/TOF analyses and online H/D exchange studies. The structures were supported by appropriate mechanistic explanation. The strategy involving use of LC and LC-MS for the identification and characterization of minute quantities of degradation products was applied on a model drug, valsartan. Three degradation products were successfully characterised without their isolation from the reaction matrix in pure form. The structures were supported by appropriate mechanistic explanation.     

Structural identification and characterization of potential impurities of pantoprazole sodium

Six impurities in pantoprazole sodium bulk drug substance were detected by a simple high performance liquid chromatographic method (HPLC) whose area percentage ranged from approximately 0.05 to 0.34%. Liquid chromatography-mass spectrometry (LC-MS) was performed to identify the molecular weight of the impurities. A thorough study was undertaken to characterize these impurities. These impurities were synthesized, subsequently characterized and were co-injected with the sample containing impurities and found the retention time match of the spiked impurities. Based on their spectral data (IR, NMR and MS), these impurities were characterized as; 5-(difluoromethoxy)-2-[[(3,4-dimethoxy-2-pyridinyl)methyl]thio]-1H-benzimidazole (Impurity-I); 5-(difluoromethoxy)-2-[[(3,4-dimethoxy-2-pyridinyl)methyl]sulfonyl]-1H-benzimidazole (Impurity-II); 5-(difluoromethoxy)-2-[[(3,4-dimethoxy-1-oxide-2-pyridinyl)methyl]sulfonyl]-1H-benzimidazole (Impurity-III); 5-(difluoromethoxy)-2-[[(3,4-dimethoxy-2-pyridinyl)methyl]thio]-1-((3,4-dimethoxy-2-pyridinyl)methyl)-1H-benzimidazole (Impurity-IV); 5-(difluoromethoxy)-2-[[(3,4-dimethoxy-2-pyridinyl)methyl]sulfinyl]-1-((3,4-dimethoxy-2-pyridinyl)methyl)-1H-benzimidazole (Impurity-V); 5-(difluoromethoxy)-2-[[(3,4-dimethoxy-1-oxide-2-pyridinyl)methyl]sulfinyl]-1H-benzimidazole (Impurity-VI). The formation of these impurities was proposed. The structure of the Impurity-II was unambiguously confirmed by single crystal X-ray diffraction (XRD) studies.     

Structural elucidation of process-related impurities in escitalopram by LC/ESI-MS and NMR

Three impurities were detected in escitalopram bulk drug by HPLC–UV and LC/MS. These impurities were marked as ESC-I, -II and -III. Two of these impurities (ESC-II and -III) were unknown and have not been reported previously. Ion trap and Q-TOF mass analyzer were employed to carry out MS/MS and accurate mass analysis of these unknown impurities. Based on mass spectrometric data and synthetic specifics the structures of ESC-II and -III were proposed as N-(chloromethyl)-3-[5-cyano-1-(4-fluorophenyl)-1,3-dihydro-2-benzofuran-1-yl]-N,N-dimethylpropan-1-aminium and N-(chloromethyl)-4-[4-cyano-2-(hydroxymethyl)phenyl]-4-(4-fluorophenyl)-4-hydroxy-N,N-dimethylbutan-1-aminium respectively. The impurities were isolated by semi-preparative HPLC and structures were confirmed by NMR spectroscopy. The plausible mechanism for the formation of impurities is also discussed.     

Identification of the degradation product of ezlopitant,a non-peptidic substance P antagonist receptor,by hydrogen deuterium exchange,electrospray ionization tandem mass spectrometry (ESI/MS/MS) and nuclear magnetic resonance (NMR) spectroscopy

The degradation product of ezlopitant was isolated from low specific activity material and identified by solution phase hydrogen/deuterium (H/D) exchange and electrospray ionization tandem mass spectrometry (ESI/MS/MS) to be an isopropyl peroxide analog of ezlopitant. The structure of the degradant was further confirmed by nuclear magnetic resonance (NMR) spectroscopy utilizing complete ¹H and ¹³C assignments. Studies were also performed to identify the factors responsible for the oxidative degradation of ezlopitant, which included salt form, storage conditions and salt formation solvent. Of all the variable studies over a 3 weeks period, only a change in the salt form prevented this oxidative degradation.     

Estimation of impurity profiles of drugs and related materials: part XXI. HPLC/UV/MS study of the impurity profile of ethynodiol diacetate

The impurity profile of ethynodiol diacetate was investigated using the HPLC/UV/MS method. Using the slightly modified HPLC method of USP 24 two impurities, earlier isolated by preparative HPLC and investigated by NMR spectroscopy were separated and characterised. The mass spectra amended by the diode-array UV spectra supported the earlier found structures (E and Z isomers of 17-ethinyl-estr-4-ene-3β,17-diol-3-acetate-17-(3′-acetoxy-2′-butenoate). Another, hitherto not described impurity, 17-ethinyl-estr-4-ene-3β,17-diol-3-acetate-17-(3-oxo-butanoate) has also been separated and characterised by means of its mass spectrum, NMR and UV spectra.     

Impact of different tissue-simulating hydrogel compartments on in vitro release and distribution from drug-eluting stents

In vitro drug release testing is an appropriate approach to identify critical parameters helping to predict drug release from drug-eluting stents (DES) prior to studying drug release behavior under in vivo conditions. Drug release and distribution from DES coated with a fluorescent model substance were studied in vitro using the vessel-simulating flow-through cell equipped with different long-term stable hydrogel compartments composed of agarose, polyacrylamide or poly(vinyl alcohol). The obtained experimental results were compared with the results of finite-element modeling obtained using experimentally determined diffusion coefficients and partition coefficients. In spite of differences regarding these parameters, experimental and mathematical data yielded only minor differences between the different gels regarding the release and distribution behavior and reasonable agreement between the modeling and the experiment was obtained. In an attempt to further elucidate the dosage form behavior, the diffusion coefficients in the gel as well as in the stent coating were systematically varied in the finite-element model. Changes in the diffusivity in the stent coating mainly impacted on the initial concentrations. Slower diffusion inside the hydrogel yielded a retarded elution from the stent coating and a higher model substance accumulation in the gel compartment at late time points.     

管花蒲公英中抗肿瘤活性化合物的快速筛选研究

目的:应用肿瘤细胞提取和色谱联用分析技术快速预测管花蒲公英中潜在的抗肿瘤活性化合物。方法:使用管花蒲公英醇提取物与人子宫颈癌细胞株(Hela)以及人胃腺癌细胞株(MK-1)孵育培养,HPLC-ESI-MS/MS及NMR技术分析和鉴定肿瘤细胞破碎液中化合物。结果:Hela细胞破碎液中检测到10个活性化合物,MK-1细胞破碎液中检测到5个活性化合物。结论:活性化合物可特异性地与靶细胞膜结合或进入靶细胞内,采用肿瘤细胞提取结合色谱联用分析技术,发现管花蒲公英中10个化合物(没食子酸,咖啡酰基酒石酸,绿原酸,4-O-咖啡酰基奎宁酸,槲皮素-3-O-α-D-吡喃阿拉伯糖苷,槲皮素-3-O-β-D-吡喃葡萄糖苷,槲皮素-3-O-α-D-呋喃阿拉伯糖苷,槲皮素-3-O-α-L-鼠李糖苷,槲皮素,木犀草素)可与Hela细胞结合,5个化合物(没食子酸,没食子酸甲酯,咖啡酸,槲皮素,木犀草素)可与MK-1细胞结合,该研究方法可预测中药提取中潜在的抗肿瘤活性成分。     

LC/MS分析加替沙星的光降解产物

目的分析加替沙星的光降解产物。方法用液相色谱仪和液质联用仪进行分离和分析。结果确证加替沙星在光照后有降解产物产生。结论加替沙星应尽量避光使用，以保证产品的质量。     

Acyclovir chemical kinetics with the discovery and identification of newly reported degradants and degradation pathways involving formaldehyde as a degradant and reactant intermediate

The purpose of this research was to determine acyclovir (ACV) acidic degradation kinetics which is relevant to gastric retentive device product design. A stability-indicating method revealed two unknown degradation products which have been identified by mass spectrometry as ACV and guanine formaldehyde adducts. In addition to the formation of these adducts, a proposed degradation scheme identifies the formation of methyl acetal ethylene glycol, formaldehyde, ethylene glycol, and guanine as additional ACV degradation products. pH-rate profiles were explained by using a rate law which assumed acid-catalyzed hydrolysis of protonated and unprotonated ACV. The predicted and observed rate constants were in good agreement. Data-driven excipient selection recommendations were based on the chemical kinetic study results, degradation scheme, and pH-rate profiles. The average activation energy for the degradation reaction was determined to be 31.3 ± 1.6 kcal/mol. The predicted ACV t_90% at 37 °C and pH 1.2 was calculated to be 7.2 days. As a first approximation, this suggests that ACV gastric retentive devices designed to deliver drug for 7 days should have acceptable drug product stability in the stomach.     

Structural elucidation of a PDE-5 inhibitor detected as an adulterant in a health supplement

A PDE-5 inhibitor was detected and isolated from a health supplement claimed to be a preparation of fresh oyster extracts and be able to promote and support healthy sexual function and endurance, etc. The structure of this PDE-5 inhibitor was elucidated using LC-UV, LC-TOF-MS, MS-MS, IR spectroscopy, and 2D NMR. It was characterized as 8-(2-(4-(hydroxymethyl)piperidin-1-yl)benzylamino)-3-ethyl-1H-imidazo[4,5-g]quinazoline-2(3H)-thione, a compound reported to be a PDE-5 inhibitor.     

Alternariol induce toxicity via cell death and mitochondrial damage on Caco-2 cells

Alternariol (AOH), a mycotoxin produced by Alternaria sp, appears as food contaminant in fruit, vegetables and cereal products. Its toxicity has been demonstrated, but the mechanisms involved have not been elucidated yet. In this study, the pathways triggered by AOH and degradation products generated on Caco-2 cells were evaluated. Cells were exposed to AOH sub-cytotoxic concentrations of 15, 30 and 60 μM. Cell cycle disruption, the induction of apoptosis/necrosis and changes in mitochondrial membrane potential (Δψm) after 24 and 48 h was asses by flow cytometry. Also, AOH and its degradation products were evaluated after 24 and 48 h by high-performance liquid chromatography with tandem mass spectrometric (LC-MS/MS) to detect and quantify its levels. Cell cycle was significantly decreased at G1 phase and increased at S and G2/M phase at the time of exposure. AOH induced necrosis, apoptosis/necrosis and loss of Δψm in a dose and time-dependent manner. The concentrations of AOH quantified in the culture media exposed to AOH decreased as the exposure time was increased. In conclusion, AOH caused cytotoxic effects supported by blocking cell cycle, decreasing cell proliferation and increasing apoptosis/necrosis cells.