在线二维液相色谱-四极杆飞行时间质谱法检测头孢噻吩钠的杂质谱

建立了在线二维液相色谱-四极杆飞行时间质谱检测头孢噻吩钠杂质谱的方法,有效地解决了流动相中含不挥发性磷酸盐的色谱系统不适合用于液相色谱-质谱快速鉴定杂质的难题。一维高效液相色谱(HPLC)以Symmetry C18为色谱柱,以磷酸盐缓冲液(pH 2.5)和乙腈梯度洗脱;二维以ACQUITY UPLC BEH C18为色谱柱,以0.1%(v/v)甲酸水溶液和0.1%(v/v)甲酸乙腈溶液梯度洗脱。以HLB C18为捕集柱,用0.1%(v/v)甲酸水溶液进行捕集和脱盐,采用正离子模式采集数据。对头孢噻吩钠中6个杂质进行了结构鉴定,对其来源进行了分析,并进一步确证了《中国药典》2010年版对头孢噻吩钠杂质A认定有误。采用本方法可以快速、简便、灵敏地对头孢噻吩钠杂质谱进行检测。     

高效液相色谱-四极杆/飞行时间质谱鉴定阿莫西林克拉维酸钾干混悬剂中的未知组分

采用高效液相色谱-四极杆/飞行时间质谱(HPLC-QTOF/MS)建立了阿莫西林克拉维酸钾混悬剂(ACPS)中未知组分的鉴定方法,以BDS Hypersil C18(4.6 mm×250 mm,5μm)为色谱柱,以乙腈-水(0.1%甲酸)为流动相,流速为1 mL/min。比较了中国药典方法和HPLC-QTOF/MS方法的相关性;利用半制备液相色谱收集未知组分,并在HPLC-QTOF/MS体系中进行定位和确认,确定未知组分在液质体系中的出峰位置和准确分子量;筛查混合空白辅料成分,并通过与标准品比对,利用HPLC-QTOF/MS鉴定ACPS的未知组分为糖精钠。方法可为抗生素药品中未知组分的鉴定提供参考。     

高效液相色谱-质谱联用分析洛伐他丁中的杂质

利用高效液相色谱-二极管阵列检测器-质谱联用方法对洛伐他丁及其杂质成分进行分离分析和结构鉴定。实验采用D iamonsil C18(5μm,4.6 mm×250 mm)为分离柱,乙腈-水(含0.1%乙酸)(65∶35)为流动相,分离并检测了洛伐他丁及其杂质;通过与DAD检测器和离子阱质谱联用,获得了它们的紫外光谱和质谱数据;紫外光谱表明除氢化洛伐他丁外其余杂质与洛伐他丁基本结构相同,利用MS和MS2数据确定了杂质的分子量和侧链结构,由此鉴定了其中10个杂质的结构。实验结果表明,高效液相色谱-二极管阵列检测器-质谱联用技术可以快速鉴定洛伐他丁中的杂质化学成分。     

气相色谱-四极杆飞行时间串联质谱在加拿大一枝黄花挥发性成分检测中的应用

采用气相色谱-四极杆飞行时间串联质谱(GC-QTOF MS)对加拿大一枝黄花中的挥发性成分进行检测。利用气相色谱的保留指数、四极杆飞行时间串联质谱的高分辨多级质谱数据以及谱库检索等对该植物的叶、茎、根三个部位中的挥发性成分进行结构鉴定。共鉴别出挥发性成分54种,其中叶47种、茎35种、根32种。主要成分均为蒎烯、表双环倍半水芹烯、柠檬烯等萜烯类化合物。结果表明,气相色谱的高分离度和四极杆飞行时间串联质谱的高分辨率可以有效地对复杂样品中的挥发性成分进行检测,结合多维数据定性技术能够显著提高未知成分鉴别的准确性。     

二维超高效液相色谱-四极杆/飞行时间质谱法解析替考拉宁杂质

建立了二维超高效液相色谱-四极杆/飞行时间质谱法（2D-UPLC-Q/TOF-MS）对替考拉宁组分分离和杂质结构解析的分析方法,有效地解决了流动相中含不挥发性磷酸盐的色谱系统不适用于液相色谱-质谱快速鉴定替考拉宁杂质的难题。一维超高效液相色谱以Octadecyl silica （ODS） hypersil色谱柱（250 mm×4.6 mm, 5 μm）进行色谱分离,以3.0 g/L磷酸二氢钠溶液（pH 6.0）/乙腈=9/1 （v/v）为流动相A、3.0 g/L磷酸二氢钠溶液（pH 6.0）/乙腈=3/7 （v/v）为流动相B进行梯度洗脱;二维超高效液相色谱以Waters ACQUITY UPLC BEH C₁₈色谱柱（50 mm×2.1 mm, 1.7 μm）进行脱盐,以0.01 mol/L甲酸铵（pH 6.0）和乙腈为流动相进行梯度脱盐洗脱。质谱在电喷雾离子源、正离子模式下,采用全信息串联质谱（MS^E）模式采集质谱数据,锥孔气流速50 L/h,锥孔电压60 V,离子源温度120 ℃,雾化气流速900 L/h,雾化气温度500 ℃,毛细管电压2500 V,碰撞能量20~50 eV。根据杂质精确质量数及其二级质谱信息推导其结构,并对替考拉宁主要成分TA_2-2的裂解规律进行了推导,发现了2个母核特征离子;对《欧洲药典》10.0收录的10个组分及22个杂质组分进行二级质谱分析,发现了3个新杂质组分。采用该法既可以使用一维超高效液相色谱根据相对保留时间进行组分准确定位,也可以使用二维超高效液相色谱-四极杆/飞行时间质谱二级质谱信息快速、简便、灵敏地对杂质进行结构鉴定,为替考拉宁的质量控制和工艺优化提供了一种新思路。     

超高效液相色谱-四极杆飞行时间质谱法快速筛查水产品中15种碱性合成色素

建立了超高效液相色谱-四极杆飞行时间质谱(UPLC-Q-TOF MS)快速检测水产品中15种碱性合成色素的方法。水产样品经乙腈(含10%(v/v)乙酸)提取,采用C₁₈复合硅胶基质分散管进行样品净化处理。目标物采用C₁₈色谱柱分离,以乙腈和0.1%(v/v)甲酸-5 mmol/L乙酸铵溶液为流动相,梯度洗脱,四极杆飞行时间串联质谱电喷雾正离子模式检测。结果表明:15种碱性色素的定量限(LOQs, S/N=10)为0.1~100 μg/kg,并在各自的线性范围内线性关系良好,相关系数(r)≥0.993。在3个加标水平下的平均回收率为80.60%~107.37%,测定结果的相对标准偏差为3.33%~6.69%(n=6)。该方法快速、简便、灵敏度高,适用于日常水产品中15种碱性合成色素的快速筛查。     

沉香的高效液相色谱-四极杆-飞行时间质谱数字化指纹图谱研究

采用高效液相色谱-四极杆-飞行时间质谱联用(HPLC-Q-TOF-MS)技术,研究构建了一种沉香数字化色谱-质谱指纹图谱的新方法。沉香药材经乙醇提取后,采用HPLC-Q-TOF-MS测定,并同时采集HPLC-Q-TOF-MS及液相色谱-紫外数据,得到液相色谱-紫外检测(HPLC-UV)色谱图和高分辨飞行时间质谱(TOF-MS)总离子流色谱图。对色谱图中的各个色谱峰进行精确质量数识别,建立数字化指纹图谱,以精确质量数结合保留时间表征沉香中的化学成分,即为每个色谱峰给出具有唯一性的数字信息,以数字化的形式反映其化学成分,并根据精确质量及同位素推算出分子式,结合二级质谱及文献资料共鉴定出30个化学成分。该方法对沉香的每种化学成分给出了类似于身份认定的数字化信息,具有唯一性,能全面反映沉香的物质成分,可为沉香的药理、药效及质量标准研究提供科学的数据。     

基于超高压液相色谱-四极杆串联飞行时间质谱技术的烟草代谢组学分析方法

对影响烟草代谢物提取的4个关键因素(溶剂、提取方式、提取时间、提取温度)在3个水平上进行了正交设计,确立了最佳提取方法;对色谱和质谱部分的实验条件进行优化,建立了基于超高压液相色谱-四级杆串联飞行时间质谱(UHPLC-Q-TOF/MS)平台的烟草代谢组学分析方法。方法可检测到2445个色谱峰,提取及分析的重现性较好。     

超高效液相色谱-四极杆-飞行时间质谱法同时测定猪肉中多类兽药残留

建立了超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF MS)快速检测猪肉中6类33种兽药残留的分析方法。样品采用QuEChERS方法进行前处理(5%(v/v)乙酸乙腈溶液提取,C18和NH₂吸附剂净化),采用ZORBAX SB-C18色谱柱(100 mm×2.1 mm, 3.5 μm)分离,以乙腈-0.1%(v/v)甲酸水溶液(含5 mmol/L乙酸铵)为流动相,梯度洗脱,Q-TOF MS电喷雾正离子模式分析检测。在全扫描采集模式下,以准分子离子峰的峰面积定量,以化合物的色谱保留时间和精确质量数定性。在Target MS/MS采集模式下,通过碎片离子的精确质量数进一步确证化合物。33种化合物在各自的线性范围内均呈现良好的线性关系,相关系数均大于0.99, 33种化合物的定量限(S/N=10)为2.5~100 μg/kg,添加回收率为67.0%~109.0%,相对标准偏差(RSD,n=6)均不高于15.1%。该方法简便、快速、灵敏,适用于猪肉中多类兽药残留的同时检测。     

超高效液相色谱-四极杆-飞行时间质谱法快速筛查血液中10种常见毒物

建立了超高效液相色谱-四极杆-飞行时间串联质谱(UPLC-Q-TOF/MS)快速筛查血液中10种毒物的检测方法。用乙酸乙酯提取血液样品,浓缩至近干后,用甲醇溶解定容,过0.22 μ m滤膜后,直接测定。目标物经ACQUITY UPLC@BEH C18 色谱柱(100 mm×2.1 mm, 1.7 μ m)分离,以甲醇和0.1%(v/v)甲酸水溶液为流动相,梯度洗脱,四极杆飞行时间串联质谱电喷雾正离子模式检测,外标法定量。结果表明:目标物在10.0~500.0 μ g/L范围内线性关系良好,相关系数为0.9908~0.9958,检出限和定量限分别为1.0~2.0 μ g/L和4.0~8.0 μ g/L。在20.0、50.0和200.0 μ g/L 3个添加水平下的平均回收率为56.7%~83.0%, 相对标准偏差为3.6%~8.9%。利用Agilent Mass Hunter PCDL Manager软件建立常见毒物的数据库,并应用于加标样品的筛查分析。该方法能快速筛查出添加的10种常见毒物,检出率达100%,且保留时间偏差均小于0.1 min,质量偏差均小于1 mDa,同位素丰度匹配得分、同位素间距得分和MS匹配得分均大于90, MS/MS图谱匹配得分均大于70。该方法可用于法庭与临床毒物分析。     

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.

     

Identification and characterization of the process‐related impurities in fasudil hydrochloride by hyphenated techniques using a quality by design approach

Following the underlying principles of quality by design mentioned in the ICH Q8 guidance, systematic approaches for the control of process‐related impurities have been taken in the manufacturing process of fasudil hydrochloride, a potent Rho‐kinase inhibitor and vasodilator. Three related impurities were found in fasudil hydrochloride lab samples by a newly developed RP‐HPLC with volatile mobile phase gradient elution and UV detection method. The elemental compositions of the impurities were determined by positive ESI high‐resolution TOF‐MS analysis of their [M + H]⁺ ions and their structures were identified through the elucidation of the product mass spectra obtained by a triple quadrupole mass spectrometer. The key impurity was further verified through synthesis and organic spectroscopy including NMR and IR spectroscopy. The origins of these impurities were located and the effective approaches to eliminate them were proposed based on the redesign of the synthetic conditions. The results obtained are important for quality control in the manufacture of fasudil hydrochloride bulk drug substance and injection.     

超高效液相色谱-静电场轨道阱高分辨质谱法测定硝苯地平中痕量基因毒性杂质

建立了测定硝苯地平中基因毒性杂质2、6和12的超高效液相色谱-静电场轨道阱高分辨质谱法(UHPLC-Orbitrap HRMS).样品以甲醇为溶剂,提取后直接进样分析.采用ACE EXCELTM 3 C18-AR色谱柱(150 mm×4.6 mm,3μm)分离,流动相为甲醇-0.1％甲酸水(65:35,v/v),等度洗...     

Investigation of amodiaquine bulk drug impurities by liquid chromatography/ion trap mass spectrometry

Three unknown impurities in an amodiaquine bulk drug sample were detected by reversed-phase high-performance liquid chromatography with ultraviolet detection (HPLC/UV). A liquid chromatography/tandem mass spectrometry (LC/MS(n)) method is described for the investigation of these impurities. Mass spectral data were acquired on an LCQ ion trap mass analyzer equipped with an electrospray ionization (ESI) source operated in positive ion mode. The fragmentation behavior of amodiaquine and its impurities has been studied. Based on the mass spectral data and the specifics of the synthetic route, the possible structures of these impurities were elucidated as 4-[(5-chloroquinolin-4-yl)amino]-2-(diethylaminomethyl)phenol (impurity I), 4-[(7-chloroquinolin-4-yl)-amino]phenol (impurity II) and 4-[(7-chloroquinolin-4-yl)amino]-2-(diethylaminomethyl)-N(1)-oxy]phenol (impurity III). The structures were confirmed by their independent synthesis and NMR spectral assignment.     

超高效液相色谱-串联四极杆飞行时间质谱和超高效液相色谱-串联三重四极杆质谱用于血浆中苦杏仁苷及其代谢产物野黑樱苷的定性和定量分析

建立了大鼠灌胃麻杏石甘汤后血浆中苦杏仁苷、野黑樱苷的定性及定量方法。样品经液液萃取净化处理,定性采用超高效液相色谱-串联四极杆飞行时间质谱仪（UPLC-QTOF-MS/MS）,经Shim-pack XR-ODS Ⅲ色谱柱（75 mm×2.0 mm,1.6 μm）分离,定量采用超高效液相色谱-串联三重四极杆质谱仪（UPLC-Q-TRAP-MS）,经Agilent C₁₈色谱柱（50 mm×2.1 mm,1.7 μm）分离,电喷雾负离子化（ESI）及MRM模式测定,流动相均为乙腈-0.1%（v/v）甲酸水溶液。结果显示苦杏仁苷、野黑樱苷在相应浓度范围内线性关系良好（相关系数分别为0.9990、0.9970）,精密度（RSD）小于9.20%,回收率为82.33%~95.25%,检出限（LOD）约为0.50 ng/mL。本方法快速简便,为血浆样品中苦杏仁苷、野黑樱苷的定性和定量分析提供良好参考。     

Liquid chromatography-tandem mass spectrometry for the identification of impurities in d-allethrin samples

A previous GC/MS study highlighting the impurity profile of the synthetic pesticide d-allethrin is extended here to validate and confirm the impurities identity through the development of soft ionisation HPLC-MS methods. To accomplish this, we developed a reverse phase LC-MS analysis in gradient elution with two distinct soft ionisation techniques, the atmospheric pressure ionisation with electrospray source (API-ESI) and the chemical ionisation (APCI). A single quadrupole and an ion trap, which allowed the simultaneous determination of the molecular masses and structural information of the impurities by acquisition of collisionally induced (CID) product ions spectrum and in-source fragmentation, were employed as analysers. Single quadrupole and ion trap analysers resulted perfectly matching in the d-allethrin impurity fragmentation patterns. All the main impurities over 0.1% identified by GC/MS were confirmed. Results indicate that the proposed HPLC/MS method was found appropriate to confirm the presence of impurities such as chrysolactone, chloro allethrin derivatives, allethrolone and chrysanthemic acid, excluding their formation under GC/MS strong ionisation condition.     

米格列奈及其3个异构体杂质的反相液相色谱分离及质谱碎裂分析

建立了超高效反相液相色谱-高分辨质谱方法以实现米格列奈及其3种异构体杂质的分离，以ACQUITY UPLC HSS T3（100 mm×2.1 mm，1.8 μ m）为色谱柱，以水-乙腈-正戊醇（75：25：1）（用甲酸调节pH至1.8）为流动相，流速为0.4 mL/min。根据Q Exactive四极杆/静电场轨道阱高分辨质谱的精确质量数及碎裂情况，发现了米格列奈及3种异构体存在碎片离子丰度的明显差异，确认其中两种为本次新发现的异构体杂质，并推断了米格列奈及3种异构体杂质可能的质谱裂解机理。经验证，该方法的灵敏度、重复性及线性均满足分析要求。在此基础上，对米格列奈异构体杂质的来源进行了探讨，发现异构体杂质1可在高温下降解产生，并对各企业的米格列奈钙原料样品进行了测定。     

Quantitation of a de‐fluorinated analogue of Casopitant Mesylate by normal‐phase liquid chromatography/mass spectrometry

The introduction of Quality by Design (QbD) in Drug Development has resulted in a greater emphasis on chemical process understanding, in particular on the origin and fate of impurities. Therefore, the identification and quantitation of low level impurities in new Active Pharmaceutical Ingredients (APIs) play a crucial role in project progression and this has created a greater need for sensitive and selective analytical methodology. Consequently, scientists are constantly challenged to look for new applications of traditional analytical techniques. In this context a normal‐phase liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS) method was developed to determine the amount of a de‐fluorinated analogue impurity in Casopitant Mesylate, a new API under development in GlaxoSmithKline, Verona. Normal‐phase LC provided the selectivity needed between our target analyte and Casopitant, while a single quadrupole mass spectrometer was used to ensure the sensitivity needed to detect the impurity at <0.05%w/w. Standard solutions and samples were prepared in heptane/ethanol (50:50, v/v) containing 1% of 2 M NH₃ in ethanol; the mobile phase consisted of heptane/ethanol (95:5, v/v) with isocratic elution (flow rate: 1.0 mL/min, total run time: 23 min). To allow the formation of ions in solutions under normal‐phase (apolar) conditions, a post‐column infusion of a solution of 0.1% v/v of formic acid in methanol was applied (flow rate: 200 µL/min). The analysis was carried out in positive ion mode, monitoring the impurity by single ion monitoring (SIM). The method was fully validated and its applicability was demonstrated by the analysis of real‐life samples. This work is an example of the need for selective and accurate methodology during the development of a new chemical entity in order to develop an appropriate control strategy for impurities to ultimately ensure patient safety. Copyright © 2010 John Wiley & Sons, Ltd.