Modern analytical methods & techniques in quality control of drugs 第十六章 药品质量控制中的 现代分析方法与技术 Modern analytical methods & techniques in quality control of drugs

第一节 概况 现代分析方法与技术，为药学的发展提供了适时而有效的手段与动力。 色谱及其联用技术： 药学研究－－分子水平。 第一节 概况 现代分析方法与技术，为药学的发展提供了适时而有效的手段与动力。 色谱及其联用技术： 药学研究－－分子水平。 手性分析： 毛细管电泳及手性色谱技术－－药物研究与质量控制提供了保障。 现代光谱技术： 药物结构鉴定， 微量杂质检定。

Capillary electrophoresis,CE 药物现代色谱法及其应用 Modern chromatogr & its application Capillary electrophoresis,CE

UPLC UltraPerformance LC® (UPLC® ) technology starts with unique 1.7 µm small-particle chemistries. Chromatographers no longer need to choose between speed and resolution—with UPLC you get both.

现代光谱法及其应用 Modern spectroscopy & its application in pharmaceutical analysis Mass spectroscopy MS Nuclear magnetic resonance spectrometry NMR X-ray diffraction method Near infrared spectrometry NIRS

Hyphenated Techniques in Chromatography 现代联用技术及其应用 Hyphenated Techniques in Chromatography GC-FTIR GC-MS UPLC-MS HPLC-NMR HPLC-MS CE-MS

第二节 液质联用技术与应用 质量分析器 检测离子 离子源 离子识别 离子检测 样品与溶剂脱 离及电离 EI ESI APCI LC/MS + 样品与溶剂脱 离及电离 EI ESI APCI LC/MS 接口 离子源 质量分析器 检测离子 HPLC 数据系统 质谱 离子识别 Quadrapole Time of Flight Fourier Transform … 离子检测 -

2.1 离子化方式

2.2 离子分离与测定模式

Full-Scan Mass Spectrometry Advantage Provides MW Information

Full-Scan MS of Buspirone (M+H)+ 150 200 250 300 350 400 450 500 m/z 25 50 75 100 Relative Abundance 386 408 Buspirone (丁螺环酮) C21H31N5O2 MW = 385 This is the full-scan MS of buspirone, which yield ions characteristic of its molecular weight. (M+Na)+

Single Ion Monitoring (SIM) Pass All Fixed m/z Pass All Advantages Targeted Analyte Monitoring High Duty Cycle Simple Disadvantages Can suffer from interferences Not as sensitive or selective as SRM (see below) SIM passes ions of one m/z essentially 100% of the time. Hence, SIM has a high duty cycle and therefore high sensitivity relative to a lower duty cycle scan mode (e.g., full-scan MS).

Product Ion Scanning: A Tandem MS Method Fixed m/z Pass All Scanning Q1 Q2 Q3 Product Ion Spectrum Product ion scan mode is used to identify fragment ions indicative of the analyte. As a scanning technique (Q3 is scanned), this mode of operation has a low duty cycle (decreased sensitivity). Advantage Provides Structural Information Disadvantage Low duty cycle

Product Ion Spectrum of Buspirone 100 150 200 250 300 350 400 m/z 25 50 75 Relative Abundance 122 386 222 265 180 (M+H)+ Here is the product ion spectrum of buspirone highlighting some of the structures of the fragment ions. Scan modes below will focus on the m/z 122 fragment ion of buspirone

Precursor Ion Spectrum Precursor Ion Scanning Scanning Pass All Fixed m/z Q1 Q2 Q3 Precursor Ion Spectrum Precursor ion scanning is the “mirror” image of product ion scan mode, whereby Q1 is scanned instead of Q3. This technique is useful in identifying different compounds that fragment to give one specific fragment ion (selected with Q3). A good example of this case is for drug metabolites. Again, since this is a scanning technique, the duty cycle is low (decreased sensitivity). Advantage ID compounds producing specific fragment ion (e.g., PO3− for phosphopeptides) Disadvantage Low duty cycle

Precursor Ion Scan Mode for Buspirone Metabolites 9 10 11 12 13 14 15 16 Time (min) 25 50 75 100 Relative Abundance 11.62 13.84 13.16 14.40 12.13 10.45 15.45 100 200 300 400 500 m/z Relative Abundance 402 386 Since buspirone fragments to generate a strong m/z 122 ion (see “Product Ion Spectrum of Buspirone” slide above), an LC/MS/MS method using precursor ion scanning was employed to identify buspirone metabolites that also fragment to give a m/z 122 ion. From the chromatogram above, the precursor ion spectrum at 11.62 minutes yields an ion at m/z 402. This means that a compound with a molecular weight of 401 was ionized to give an (M+H)+ at m/z 402, which was selected with Q1, fragmented in Q2 and passed through Q3, which was fixed at m/z 122. When compared to buspirone, which eluted at 15.45 minutes, the mass difference is 16 daltons, indicating that this metabolite is hydroxylated. Precursor Ion Scan: Q3 set to m/z 122

Neutral Loss Scanning Advantage Disadvantage Q1 Q3 Q2 Linked Pass All Scanning Scanning Q1 Q3 Q2 Neutral Loss Spectrum Neutral loss scan mode is unique in that both Q1 and Q3 scan simultaneously. However, they do not transmit ions of the same m/z at the same time; rather, Q1 passes ions that are higher in m/z than Q3 at a given time. The difference in m/z equates to a neutral loss, which can be recorded as a neutral loss spectrum. This scan mode is useful for identifying drug metabolites, such as glucuronide conjugates (i.e., addition of a sugar group to a drug molecule). In the case of a glucuronidated drug, Q1 would pass ions with a m/z of (M + 176)+ while at the same time Q3 passes ions of m/z of (M)+, since the glucuronide would be lost during fragmentation in Q2. Advantage Screen for compounds producing specific neutral loss (e.g., loss of 176 for glucuronide conjugates) Disadvantage Low duty cycle

Neutral Loss Scan of Buspirone Metabolites 9 10 11 12 13 14 15 16 Time (min) 25 50 75 100 Relative Abundance 13.92 11.69 13.21 15.50 10.58 100 200 300 400 500 m/z Relative Abundance 402 386 Since buspirone fragments to generate a m/z 265 ion (see “Product Ion Spectrum of Buspirone” slide above), the corresponding neutral loss is 121 (m/z 386 – m/z 265). Setting the TSQ Quantum to neutral loss mode for 121, there are several metabolites that are identified. The peak at 13.92 minutes yielded a neutral loss spectrum (top right) where the base peak ion is m/z 402. Since this is 16 daltons higher than the unmodified buspirone (15.50 min), this compound also corresponds to a hydroxylated metabolite. Neutral Loss Scan: Q1/Q3 difference set to 121 Da

Selected Reaction Monitoring (SRM) Fixed m/z Pass All Q1 Q2 Q3 Advantages Targeted Analyte Monitoring High Duty Cycle “Simultaneous” Monitoring of Multiple Transitions Disadvantage No “advanced” structural information This is the scan mode mainly used for quantitative LC/MS/MS experiments on a triple quadrupole mass spectrometer. This is due to the high duty cycle of this scan mode (nearly 100%), which yields high sensitivity, and the high selectivity of SRM, which only transmits ions of a specific parent m/z and product m/z.

MS/MS Selectivity in Complex Matrices This slide demonstrates the importance of selectivity afforded the triple quad using SRM. An analyte, astemizole, spiked into rat plasma at a concentration of 1.5 ng/mL was analyzed using SRM and is easily detected (middle chromatogram). However, the same sample analyzed in SIM mode, which only measures a parent m/z, could not be observed due to the presence of high chemical background at the same m/z. This is the main reason triple quads are favored over single quads, which can only operate in SIM mode, for trace level quantitation in complex matrices. 息斯敏——阿斯咪唑(astemizole)

2.3药物分析中的典型应用 Chlroamphenicol(氯霉素，CAP)残留测定 黄杨生物碱成分鉴定 苯甲酸利扎曲普坦人体药代动力学研究

2.3.1 Chlroamphenicol(氯霉素，CAP)残留测定 C11H12Cl2N2O5 FMW=323.13 【类别】　酰胺醇类抗生素　 【适应症】本品是治疗伤寒、副伤寒的首选药物，外用可治疗沙眼。因脑脊液浓度高，故常用于治疗细菌性脑膜炎和脑脓肿。此外，尚可外用治疗痤疮、酒糟鼻、脂溢性皮炎等。 被农业养殖滥用！ 肉食品中严格检查。

Column: Thermo Hypersil Gold C18 (100×2.1 mm, 5µ) HPLC analysis was performed on the Finnigan Surveyor HPLC module with MS Pump and Autosampler Column: Thermo Hypersil Gold C18 (100×2.1 mm, 5µ) Mobile Phase: A: Water; B: Acetonitrile Column Temperature: 40 oC Gradient Program: 0.25 mL/min Injection: 20 uL with loop Time (min) % A % B 80 20 2.5 3.0 3.1 5.0

Operation Conditions for CAP Ion source: ESI Ion polarity: Negative Spray voltage: 4000 V Sheath gas pressure: 45 Auxiliary gas pressure: 15 Ion transfer capillary temperature: 300 oC Source CID: 8 V Scan Type: SRM, 3 transitions of [M-H]-(m/z: 321) (321152, 321  194 and 321  257) Q1 peak width 0.7 Da in SRM or 0.2 Da in H-SRM Q3 peak width 0.7 Da Collision Pressure: Ar at 1.3 mTorr

Q1 peak width and H-SRM experiment 0.7 Da in SRM or 0.2 Da in H-SRM Q3 peak width 0.7 Da Enabling the H-SRM experiment Highly Selective Selected Reaction Monitoring (H-SRM) Reduces “isobaric” chemical noise Increases confidence of analysis & improved LOQ

CAP SRM Result: CAP Standard Q1 peak width = 0.7 Da TIC 321->152 321->194 321->257 Peak Area Counts = 2.4E4

CAP SRM Result: Kidney Blank TIC 321->152 321->194 321->257

CAP SRM Result: Kidney Spiked (0.5ng/g) TIC 321->152 321->194 321->257 CAP CAP detected Not accurate for confirmation

CAP H-SRM Result: CAP Standard Q1 peak width = 0.2 Da TIC 321->152 321->194 321->257 Peak Area Counts = 7.3E3

CAP H-SRM Result: Kidney Blank TIC 321->152 321->194 321->257 No CAP detected

CAP H-SRM Result: Kidney Spiked (0.5ng/g) TIC 321->152 321->194 321->257

2.3.2 黄杨宁生物碱HPLC-MS联用鉴定 黄杨科植物小叶黄杨Buxus microphlla Sieb. et. Zucc. var. sinica Rehd.et Wils中含有具有较强心血管疾病治疗活性的孕甾烷生物碱，主要含环维黄杨星D、环黄杨碱D和环常绿黄杨碱C等生物碱成分。  

色谱条件 色谱柱：Lichrospher SiO2 (250mm×4.6mm,5 µm) 流动相：四氢呋喃-甲醇-乙腈-氨水 ( 32:50:13:3) 流速：1mL·min -1 柱温：30℃ ELSD参数：漂移管温度70℃ 雾化气体（N2） 流速：1.5 L·min -1 黄杨生物碱HPLC-ELSD色谱图

环维黄杨星D和有关生物碱含量测定结果 次数 环维黄杨星D含量% 峰1生物碱含量% 峰2生物碱含量% 峰4生物碱含量% 峰5生物碱含量% 有关生物碱总含量% 1 86.85 3.57 8.09 0.79 0.92 13.37 2 87.08 3.84 8.54 0.80 0.98 14.15 3 85.83 3.46 8.82 0.81 0.95 14.03 4 85.70 3.54 8.76 0.87 0.93 14.10 5 85.05 3.52 8.64 0.74 0.85 13.75 6 84.84 3.74 9.03 0.97 14.53 Mean 85.89 3.61 8.65 13.99 RSD% 1.07 3.65 3.38 4.62 4.63 2.8

环维黄杨星D及其有关生物碱的鉴别 质谱条件 电喷雾离子化正离子检测 喷口电压5000V 雾化气压35psi 辅助气压力5psi 质谱条件 电喷雾离子化正离子检测 喷口电压5000V 雾化气压35psi 辅助气压力5psi 毛细管温度350℃ 碰撞气氩气压力1.5mTorr 色谱条件 色谱柱：Lichrospher SiO2 (250mm×4.6mm,5 µm) 流动相：四氢呋喃-甲醇-乙腈-氨水 ( 32:50:13:3) 流速：1mL·min -1 柱温：30℃

6 7 黄杨宁LC-MS/MS全扫描色谱放大图 4 8 9 5 2 1 3 黄杨宁LC-MS/MS全扫描色谱图

[M+H]+=370 峰1母离子质荷比 峰1二级质谱图

可能为峰1的黄杨宁有关生物碱 Cyclobuxomicreine K Cyclobuxosuffrine K Buxenone M Cyclobuxoviridine B 可能为峰1的黄杨宁有关生物碱

[M+H]+= 431 峰2母离子质荷比 峰2二级质谱图

可能为峰2的黄杨宁有关生物碱 Cyclomicrophylline B Buxazidine B Cyclobuxoxazine C Cyclomicrophylline C 可能为峰2的黄杨宁有关生物碱

[M+H]+= 415 峰3母离子质荷比 峰3二级质谱图

可能为峰3的黄杨宁有关生物碱 Cyclokreanine B Cycloprotobuxine A Cyclovirobuxeine B 16-deoxybuxidienine C 可能为峰3的黄杨宁有关生物碱

环常绿黄杨碱C [M+H]+= 417 峰4母离子质荷比 峰4二级质谱图

[M+H]+= 401 383.34 峰5母离子质荷比 峰5二级质谱图

可能为峰5的黄杨宁有关生物碱 Cycloprotobuxine C Buxaminol E Buxocyclamine A Cyclobuxine B 可能为峰5的黄杨宁有关生物碱

环黄杨碱D [M+H]+= 387 峰6母离子质荷比 峰6二级质谱图

环维黄杨星D [M+H]+= 403 峰7母离子质荷比 峰7二级质谱图

[M+H]+= 375 峰8母离子质荷比

371.17 357.15 峰8二级质谱图 峰9二级质谱图

m/z=357 m/z=375 m/z=326 m/z=344 357.15 m/z=309

[M+H]+= 389 峰9母离子质荷比

371.17 峰9二级质谱图

m/z=371 m/z=389 m/z=340 m/z=358 371.17 m/z=309

HPLC-ELSD法黄杨宁有关生物碱归属表 峰位号 tR (min) [M+H]+ (m/z) 特征碎片离子 可能生物碱名称 1 3.92 370 339，325，283， 135，70，58 Cyclobuxomicreine K, Cyclobuxosuffrine K, Buxenone M, Cyclobuxoviridine B 2 4.03 431 413，382，323， 86，70，58 Buxazidine B, Cyclomicrophylline B, Cyclobuxoxazine C, Cyclomicrophylline C 3 5.35 415 384，84，58 Cycloprotobuxine A,Cyclokreanine B, Cyclovirobuxeine B, 16-deoxybuxidienine C 4 5.92 417 399，386，368， 84，58 Cylcyclovirobuxine C 5 6.11 401 370，352，326， 171，58 Cycloprotobuxine C, Buxaminol E, Buxocyclamine A, Cyclobuxine B 6 7.03 387 369，356，338， Cyclobuxine D 7 7.63 403 385，372，354， 70，58 Cyclovirobuxine D 8 8.28 375 357，344，326， 309，58 未有相关文献报道 9 9.77 389 371，358，340， 173，70，58 可能为Cyclobuxine D双键加氢还原产物

苯甲酸利扎曲普坦 (Rizatriptan Benzoate) 2.3.3 苯甲酸利扎曲普坦人体药代动力学研究 苯甲酸利扎曲普坦 (Rizatriptan Benzoate) MW: 391.47 分子式：C15H19O5·C7H6O2 5-HT受体拮抗剂

药理作用 刺激大脑血管壁的后接点5-HT1B受体收缩血管，降低颅内血管通透性； 刺激三叉神经前突触5-HT1D受体，调节神经递质的释放，抑制硬膜的神经原性炎症反应和血浆外渗； 阻止血管肽的释放，使血管口径正常化，通过收缩颅内血管并抑制神经炎症； 刺激脑干5-HT1B或5-HT1D受体，抑制三叉神经核兴奋； 减少颈动脉血流； 透过血脑屏障，增加脑血流量。

实验内容 建立苯甲酸利扎曲普坦在血浆、尿样浓度的LC-MS/MS测定方法 苯甲酸利扎曲普坦分散片和胶囊进行生物等效性试验 苯甲酸利扎曲普坦片体内药代动力学研究  单剂量 (5,10,15 mg)  多次给药(10 mg)  稳态(10 mg)

LC-MS/MS测定方法建立 测定方法选择 LC-MS/MS 质谱条件优化 色谱条件选择 m/z 270m/z 158  色谱柱选择  缓冲盐选择 血浆处理方法  液液萃取法  蛋白沉淀法 内标选择 LC-MS/MS m/z 270m/z 158 Phenomenx PFP 1%冰醋酸, 0.2%醋酸铵 蛋白沉淀法 盐酸曲马多

色谱条件 流动相A：醋酸盐缓冲液(1%冰醋酸，2%醋酸铵;pH 3.5) 流动相B：甲醇(0.1%甲酸) 梯度条件过程：0 min (B50%)→1.0 min (B95%)→4.5 min (B95%)→4.6 min (B50%) →6.5 min (B50%)

空白溶剂色谱图 预处理的空白血浆色谱图

质谱条件 离子检测方式： ESI+ SRM 检测对象： 利扎曲普坦 m/z 269.9→158.1 曲马多 m/z 263.9→58.0 喷口电压： 5000 V 雾化气压： 35 psi 辅助气压力： 5 psi 毛细管温度： 350℃ 碰撞气氩气压力： 1.3mTorr 碰撞能量： 20 eV

质谱条件优化 利扎曲普坦LC-MS质谱扫描图（m/z =270）

利扎曲普坦LC-MS/MS质谱扫描图（m/z=158）

利扎曲普坦质谱裂解图 m/z 270m/z 158 （碰撞能量：20ev）

缓冲盐条件(0.1%HCOOH,v/v; 0.2%醋酸铵,w/v; pH3.0） 色谱条件选择 缓冲盐条件(0.1%HCOOH,v/v; 0.2%醋酸铵,w/v; pH3.0） Intersil-ODS C18 Lichrospher-C18 Zorbak-ODS C18 Lichrospher-CN Phenomenx Curosil-PFP 色谱柱 采用PFP（五氟苯基）柱，样品的保留适当，峰型良好，有机相和水相的比例也较为适当。

甲醇:水=(50:50) 0.05% 0.1% 0.2% 0.2% (w/v)醋酸铵样品峰响应高，拖尾得到一定程度的改善。 醋酸铵 (w/v) 0.1% HCOOH 0.1% HAC 0.2% HAC 0.5% HAC 1.0% HAC 1%HAC水相pH值为3.5，样品保留时间合适，峰形良好。

血浆处理方法 苯甲酸利扎曲普坦血浆提取条件试验

内标选择 佐米曲普坦 (Zolmitriptan) MW：287 分子式：C16H21N3O2

盐酸曲马多 (Tramadol hydrochloric) MW：299.8 分子式：C16H25NO2

曲马多LC-MS质谱扫描图（m/z=264）

曲马多LC-MS/MS质谱扫描图（m/z=58）

曲马多质谱裂解图 m/z 264 m/z 58

单次给药 受试者单次口服苯甲酸利扎曲普坦片后，苯甲酸利扎曲普坦的Cmax和AUC与服药剂量成正比关系。 Cmax－Dose AUC－Dose

数据分析 受试者单剂量口服低中高剂量后的平均血药浓度-时间曲线

受试者单剂量口服低中高剂量后的 平均累积排泄量-时间曲线

受试者多次服药第3～6天时平均谷时血药浓度-时间曲线

受试者多次口服达稳态后的平均药时曲线

2.4 其他质谱技术与特点

不断 探索 成功来源于 创新 意识 勇于 实践