《药物分析》第十六章药品质量控制中的现代分析方法与技术pdf
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– Low duty cycle
Neutral Loss Scan of Buspirone Metabolites
100
13.92
402
11.69 75
Relative Abundance Relative Abundance
50
386
25 10.58
13.21
15.50
9
10
11
12
13
14
15
–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
Time (min)
– Can suffer from interferences
– Not as sensitive or selective as SRM (see below)
Product Ion Scanning: A Tandem MS Method
Fixed m/z
Pass All
Scanning
Q1
Q2
• Advantage
– Provides MW Information
Full-Scan MS of Buspirone
100
(M+H)+ 386
Relative Abundance
75
Buspirone
50
(丁螺环酮)
C21H31N5O2
MW = 385
25
(M+Na)+
408
150
200
250
CAP SRM Result: CAP Standard
Q1 peak width = 0.7 Da
CAP
TIC
321->152 321->194
Peak Area Counts = 2.4E4
321->257
CAP SRM Result: Kidney Blank
TIC 321->152 321->194 321->257
CAP SRM Result: Kidney Spiked (0.5ng/g)
TIC
CAP
321->152
CAP detected
321->194
321->257
Not accurate for confirmation
CAP H-SRM Result: CAP Standard
Q1 peak width = 0.2 Da
Q1 peak width and H-SRM experiment
Q1 peak width Q3 peak width
0.7 Da in SRM or 0.2 Da in H-SRM 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
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
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。 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。
• Disadvantage – Low duty cycle
Precursor Ion Scan Mode for Buspirone Metabolites
100
11.62
402
13.84 75
Relative Abundance Relative Abundance
50
386
13.16
25
10.45
Q1 peak width Q3 peak width Collision Pressure:
ESI Negative 4000 V 45 15 300 oC
8V SRM, 3 transitions of [M-H]-(m/z: 321) (321152, 321 194 and 321 257) 0.7 Da in SRM or 0.2 Da in H-SRM 0.7 Da Ar at 1.3 mTorr
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பைடு நூலகம்
3
85.83 3.46 8.82 0.81 0.95 14.03
TIC
321->152 321->194
Peak Area Counts = 7.3E3
321->257
CAP H-SRM Result: Kidney Blank
TIC
321->152 321->194
No CAP detected
321->257
CAP H-SRM Result: Kidney Spiked (0.5ng/g)
Q2
Q3
Neutral Loss Spectrum
• Advantage
– Screen for compounds producing specific neutral loss (e.g., loss of 176 for glucuronide conjugates)
• Disadvantage
– Targeted Analyte Monitoring – High Duty Cycle – “Simultaneous” Monitoring
of Multiple Transitions
• Disadvantage
– No “advanced” structural information
300
350
400
450
500
m/z
Single Ion Monitoring (SIM)
Fixed m/z
Pass All
Pass All
• Advantages
– Targeted Analyte Monitoring
– High Duty Cycle – Simple
• Disadvantages
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。
HPLC analysis was performed on the Finnigan Surveyor HPLC module with MS Pump and Autosampler
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.
CAP
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。
50
25
150
222
265
180
100
150
200
250
300
350
400
m/z
Precursor Ion Scanning
Scanning
Pass All
Fixed m/z
Q1
Q2
Q3
Precursor Ion Spectrum
• Advantage
– ID compounds producing specific fragment ion (e.g., PO3− for phosphopeptides)
环维黄杨星D和有关生物碱含量测定结果
次数 1
环维黄 峰1生 峰2生 峰4生 峰5生 有关生 杨星D 物碱含 物碱含 物碱含 物碱含 物碱总 含量% 量% 量% 量% 量% 含量%
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
12.13
14.40 15.45
9
10
11
12
13
14
15
16
100
200
300
400
500
Time (min)
m/z
Precursor Ion Scan: Q3 set to m/z 122
Neutral Loss Scanning
Scanning
Linked
Pass All
Scanning
Q1
MS/MS Selectivity 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
现代联用技术及其应用
Hyphenated Techniques in Chromatography
GC-FTIR
GC-MS
HPLC-MS
HPLC-NMR
UPLC-MS
CE-MS
第二节 液质联用技术与应用
离子源
质量分析器
+
+ ++
++ +
+ ++
- +
+
++
+ -+
离子识别
¨ Quadrapole
16
100
200
300
400
500
Time (min)
m/z
Neutral Loss Scan: Q1/Q3 difference set to 121 Da
Selected Reaction Monitoring (SRM)
Fixed m/z
Pass All
Fixed m/z
Q1
Q2
Q3
• Advantages
样品与溶剂脱 ¨ Time of Flight
离及电离 EI
¨ Fourier Transform ¨…
LC/MS ¨ ESI
接口 ¨ APCI
HPLC
检测离子
++ +
+
离子检测
质谱
数据系统
2.1 离子化方式
2.2 离子分离与测定模式
Full-Scan Mass Spectrometry
%A
%B
0
80
20
2.5
20
80
3.0
20
80
3.1
80
20
5.0
80
20
–Injection: 20 uL with loop
Operation Conditions for CAP
Ion source: Ion polarity: Spray voltage: Sheath gas pressure: Auxiliary gas pressure: Ion transfer capillary temperature: Source CID: Scan Type:
Q3
Product Ion Spectrum
• Advantage
– Provides Structural Information
• Disadvantage
– Low duty cycle
Product Ion Spectrum of Buspirone
122
100
(M+H)+
386
75
Relative Abundance
Neutral Loss Scan of Buspirone Metabolites
100
13.92
402
11.69 75
Relative Abundance Relative Abundance
50
386
25 10.58
13.21
15.50
9
10
11
12
13
14
15
–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
Time (min)
– Can suffer from interferences
– Not as sensitive or selective as SRM (see below)
Product Ion Scanning: A Tandem MS Method
Fixed m/z
Pass All
Scanning
Q1
Q2
• Advantage
– Provides MW Information
Full-Scan MS of Buspirone
100
(M+H)+ 386
Relative Abundance
75
Buspirone
50
(丁螺环酮)
C21H31N5O2
MW = 385
25
(M+Na)+
408
150
200
250
CAP SRM Result: CAP Standard
Q1 peak width = 0.7 Da
CAP
TIC
321->152 321->194
Peak Area Counts = 2.4E4
321->257
CAP SRM Result: Kidney Blank
TIC 321->152 321->194 321->257
CAP SRM Result: Kidney Spiked (0.5ng/g)
TIC
CAP
321->152
CAP detected
321->194
321->257
Not accurate for confirmation
CAP H-SRM Result: CAP Standard
Q1 peak width = 0.2 Da
Q1 peak width and H-SRM experiment
Q1 peak width Q3 peak width
0.7 Da in SRM or 0.2 Da in H-SRM 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
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
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。 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。
• Disadvantage – Low duty cycle
Precursor Ion Scan Mode for Buspirone Metabolites
100
11.62
402
13.84 75
Relative Abundance Relative Abundance
50
386
13.16
25
10.45
Q1 peak width Q3 peak width Collision Pressure:
ESI Negative 4000 V 45 15 300 oC
8V SRM, 3 transitions of [M-H]-(m/z: 321) (321152, 321 194 and 321 257) 0.7 Da in SRM or 0.2 Da in H-SRM 0.7 Da Ar at 1.3 mTorr
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பைடு நூலகம்
3
85.83 3.46 8.82 0.81 0.95 14.03
TIC
321->152 321->194
Peak Area Counts = 7.3E3
321->257
CAP H-SRM Result: Kidney Blank
TIC
321->152 321->194
No CAP detected
321->257
CAP H-SRM Result: Kidney Spiked (0.5ng/g)
Q2
Q3
Neutral Loss Spectrum
• Advantage
– Screen for compounds producing specific neutral loss (e.g., loss of 176 for glucuronide conjugates)
• Disadvantage
– Targeted Analyte Monitoring – High Duty Cycle – “Simultaneous” Monitoring
of Multiple Transitions
• Disadvantage
– No “advanced” structural information
300
350
400
450
500
m/z
Single Ion Monitoring (SIM)
Fixed m/z
Pass All
Pass All
• Advantages
– Targeted Analyte Monitoring
– High Duty Cycle – Simple
• Disadvantages
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。
HPLC analysis was performed on the Finnigan Surveyor HPLC module with MS Pump and Autosampler
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.
CAP
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。
50
25
150
222
265
180
100
150
200
250
300
350
400
m/z
Precursor Ion Scanning
Scanning
Pass All
Fixed m/z
Q1
Q2
Q3
Precursor Ion Spectrum
• Advantage
– ID compounds producing specific fragment ion (e.g., PO3− for phosphopeptides)
环维黄杨星D和有关生物碱含量测定结果
次数 1
环维黄 峰1生 峰2生 峰4生 峰5生 有关生 杨星D 物碱含 物碱含 物碱含 物碱含 物碱总 含量% 量% 量% 量% 量% 含量%
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
12.13
14.40 15.45
9
10
11
12
13
14
15
16
100
200
300
400
500
Time (min)
m/z
Precursor Ion Scan: Q3 set to m/z 122
Neutral Loss Scanning
Scanning
Linked
Pass All
Scanning
Q1
MS/MS Selectivity 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
现代联用技术及其应用
Hyphenated Techniques in Chromatography
GC-FTIR
GC-MS
HPLC-MS
HPLC-NMR
UPLC-MS
CE-MS
第二节 液质联用技术与应用
离子源
质量分析器
+
+ ++
++ +
+ ++
- +
+
++
+ -+
离子识别
¨ Quadrapole
16
100
200
300
400
500
Time (min)
m/z
Neutral Loss Scan: Q1/Q3 difference set to 121 Da
Selected Reaction Monitoring (SRM)
Fixed m/z
Pass All
Fixed m/z
Q1
Q2
Q3
• Advantages
样品与溶剂脱 ¨ Time of Flight
离及电离 EI
¨ Fourier Transform ¨…
LC/MS ¨ ESI
接口 ¨ APCI
HPLC
检测离子
++ +
+
离子检测
质谱
数据系统
2.1 离子化方式
2.2 离子分离与测定模式
Full-Scan Mass Spectrometry
%A
%B
0
80
20
2.5
20
80
3.0
20
80
3.1
80
20
5.0
80
20
–Injection: 20 uL with loop
Operation Conditions for CAP
Ion source: Ion polarity: Spray voltage: Sheath gas pressure: Auxiliary gas pressure: Ion transfer capillary temperature: Source CID: Scan Type:
Q3
Product Ion Spectrum
• Advantage
– Provides Structural Information
• Disadvantage
– Low duty cycle
Product Ion Spectrum of Buspirone
122
100
(M+H)+
386
75
Relative Abundance