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沃特世讲义3_XP最大化LC效率

2014-3-24
Agenda
行业趋势
最大程度提升HPLC效率 ——2.5 μm超高性能[XP]色谱柱
王丹丹 Dandan_wang@https://www.wendangku.net/doc/a610785217.html, 2014.3.20 长沙
制药行业应用食品行业应用柱筛选与方法开发应用注意事项
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Agenda
行业趋势
制药行业应用食品行业应用柱筛选与方法开发应用注意事项
中国用户应用实例分享 ——面临挑战
样品性质不稳定：分析时间过长：
- 每针样品分析需66分钟，第一天上午送样6批，要到第三天中午才能得到结果 - 头孢噻肟钠属beta-内酰胺类抗生素，样品溶液不稳定，要求溶液配制后尽快检测
实验室的任务多样且逐年递增：仪器和人员增长成本：
- 质量部承担了产品原料检验，生产过程中间体监测，原料药出口检验，成品出厂检验等多任务质量分析工作 - 既有品种产量逐年增加，同时不断承接新品种，测试任务逐年大幅增加
- 每年都有增加HPLC系统的购买计划，但还是感觉仪器不够用，且实验室空间有限，另外分析人员为完成分析任务除倒班外还经常加班
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中国用户应用实例分享 ——UPLC技术解决分析瓶颈
HPLC
仍然是仪器配置主体
What are the challenges? ?Need to utilize existing LC assets while improving throughput ?Method transfer between different LC platforms ?Selectivity choice for challenging separations ?Solvent cost reduction is minimal due to the requirement of 3.0 – 4.6 mm ID columns to overcome system dispersion. ?Transition strategy between different LC platforms? ?Cannot transfer regulated methods without full revalidation ?Would like to reduce backpressure when possible
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可无忧转换的HPLC柱平台
如何最大程度的利用既有HPLC系统，系统，同时确保向UPLC技术的顺利过渡升级？技术的顺利过渡升级？
提供极佳的pH耐受性
eXtended Performance [XP] 2.5 μm Columns
提供多种选择性
1.7 [UPLC], 2.5, 3.5, 5 and 10 μm
1.7 [UPLC], 2.5, 3.5 and 5 μm CSH 1.8 [UPLC], 2.5, 3.5 and 5 μm HSS
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提高产率
Tolmetin
0.30
HPLC
Diclofenac
向UPLC升级的方式
XSelect CSH C18
4.6 x 100 mm, 3.5 μm HPLC System
Naproxen
Fenoprofen
AU
0.20
0.10
0.00 0.00
4.00
8.00
12.00
16.00 Minutes
Indomethacin
3.5 μm
20.00
24.00
28.00
0.30
XSelect CSH C18 XP
4.6 x 75 mm, 2.5 μm HPLC System
AU
0.20
2.5 μm XP
2X Faster than 3.5 μm HPLC
0.10
0.00 0.00
2.00
4.00
6.00
8.00 Minutes
10.00
12.00
14.00
16.00
0.30
ACQUITY UPLC CSH C18
2.1 x 50 mm, 1.7 μm UPLC System
1.7 μm
AU
0.20
0.10
9X Faster than 3.5 μm HPLC 5X Faster than 2.5 μm HPLC
0.50 1.00 1.50 Minutes
0.00
0.00
2.10
2.50
3.00
3.50
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HPLC
提速USP方法分析克拉霉素系统上，系统上，HPLC分析柱 XP柱
系统上，分析柱柱
提速USP方法分析克拉霉素 XP柱：HPLC系统 UPLC系统
柱：系统系统
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2.5 μm eXtended Performance Columns
Packed in ultra-low dispersion hardware
– Maintains XBridge or XSelect HPLC branding
Agenda
Designed to withstand high pressure
– 4.6 mm ID capable of 9,000 PSI – 2.1 and 3.0 mm ID compatible with UPLC pressures
New 2.5 μm eXtended Performance XP Columns compatible with HPLC and UPLC Technology
行业趋势
- 直接沿用原等度条件方法 - Case Study： Study：如何依从USP<621> 如何依从USP<621>规范 USP<621>规范？规范？
制药行业应用
食品行业应用
Flexibility in configuration
– 2.1, 3.0 and 4.6 mm ID – 30, 50, 75，100 and 150mm lengths – 14 stationary phases
柱筛选与方法开发
Packed with XBridge [BEH] and XSelect [CSH and HSS] 2.5 μm particles and chemistries
– BEH C18, Shield RP18, C8, Phenyl, HILIC and Amide – CSH C18, Phenyl-Hexyl and Fluoro-Phenyl – HSS C18, T3, C18 SB, Cyano and PFP
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应用注意事项
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ChP2010方法：盐酸溴己新——系统适应性
USP
方法：噻康唑——有关物质系统适应性
Symmetry C18, 3.5um, 4.6x150mm 16.804min
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USP
通则<621>
Variable
Particle Size Column Length Flow Rate Column ID Injection Volume Column Temperature Mobile Phase pH
Compendial (USP) Method Transfer Example
Allowed Adjustments in USP Chromatography <621>
-50% ±70% ±50% Any allowed Any reduction ±10% ±0.2 unit
Compendial (USP) method transfer examples Isocratic Assay: Levonorgestrel （左炔孕）and Ethinyl Estradiol（乙炔雌二醇）
– Compare original HPLC method with:
o o o
酮
XP column methods on Alliance HPLC system XP column method on ACQUITY UPLC H-Class system ACQUITY UPLC column method on ACQUITY UPLC H-Class system
XP 2.5 μm columns fall within the allowable guidelines (within -50% allowance for 5 μm particle columns)
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USP Assay: Levonorgestrel and Ethinyl Estradiol
Isocratic HPLC Method
Column: Injection Volume: Flow Rate: Mobile Phase: Detection Wavelength: XBridge C8 4.6 x 150 mm 5 μm (L7) 50 μL 1.00 mL/min
Original Isocratic USP Assay: 4.6 x 150 mm, 5 μm (Alliance HPLC)
Flow Rate: Pressure: Run time: USP Res:
0.050
System Suitability Requirements
USP Resolution: Peak Area Precision: NLT 2.5 NMT 2.0% RSD
0.070 0.060
1.00 mL/min 1400 psi 8.00 min
levonorgestrel
System Suitability Requirements
USP Resolution: Peak Area Precision: NLT 2.5 NMT 2.0% RSD
6.9
ethinyl estradiol
35:15:45 ACN:MeOH:Water UV 230 nm
AU
0.040 0.030 0.020 0.010 0.000 0.00 1.00 2.00 3.00 4.00
5.00 Minutes
6.00
7.00
8.00
9.00
10.00
Questions Can we transfer this ‘simple’ method? What combination of column and system? What compromises (if any) must be made? What conditions provide the greatest benefits?
Remember:
L/dp = 30000 N = 1X Rs = 1X Run time = 1X Pressure = 1X
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1 L N∝ Rs ∝ N Fopt ∝ dp dp
Run Time ∝ L × 1 F
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Isocratic Method Transferred to 4.6 mm XP Columns (Alliance HPLC)
0.070 0.060 0.050
Isocratic Method Transferred to 4.6 mm XP Columns (Alliance HPLC)
Could not run at SCALED flow rate due to system pressure limitations
0.070 0.060
levonorgestrel
AU
ethinyl estradiol
AU
0.040 0.030 0.020 0.010 0.000 0.00 0.50 1.00 1.50 2.00 2.50
0.030 0.020 0.010 0.000
ethinyl estradiol
Results: Rs = 0.9X Run time = 0.5X Pressure = 2X
levonorgestrel
4.6 x 75 mm Flow Rate: Pressure: Run time: USP Res:
XP 2.5 μm 1.00 mL/min 2600 psi 4.00 min 6.5
0.050 0.040
4.6 x 75 mm Flow Rate: Pressure: Run time: USP Res:
XP 2.5 μm 1.00 mL/min 2600 psi 4.00 min 6.5
Could not run at SCALED flow rate due to system pressure limitations
Results: Rs = 0.9X Run time = 0.5X Pressure = 2X
3.00
0.060
0.050
ethinyl estradiol
levonorgestrel
0.040
levonorgestrel
4.6 x 50 mm XP 2.5 μm Flow Rate: 2.00 mL/min Pressure: 3600 psi Run time: 1.30 min USP Res: 5.0
ethinyl estradiol
3.50 4.00 4.00 Minutes
4.50
5.00
5.50
6.00
6.50
7.00
0.00
0.50
1.00
1.50
2.00
2.50
3.00
> 50% flow rate change not <621> compliant
LOWER L/dp column (20000) to reduce pressure:
0.060 0.050 0.040
4.6 x 50 mm XP 2.5 μm Flow Rate: 1.50 mL/min Pressure: 3050 psi Run time: 1.60 min USP Res: 5.2
3.50 4.00 4.00 Minutes
4.50
5.00
5.50
Both flow rates are <621> 6.00 6.50 7.00 compliant
LOWER L/dp column (20000) to reduce pressure:
AU
0.030
0.030 0.020 0.010 0.000
0.020
0.010
Results: Rs = 0.7X Run time = 0.16X Pressure = 2.9X
1.00 1.20 1.30 1.40 Minutes 1.60 1.80 2.00 2.20 2.40
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Results: Rs = 0.8X Run time = 0.2X Pressure = 2.2X
0.000 0.00
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AU
0.20
0.40
0.60
0.80
-0.010 0.00
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0.20
0.40
0.60
0.80
1.00 Minutes
1.20
1.40
1.60 1.60
1.80
2.00
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Isocratic Method Transferred to ACQUITY UPLC H-Class System
0.08
Isocratic Method Transferred to ACQUITY UPLC H-Class System
0.08
0.06
levonorgestrel
ethinyl estradiol
0.04
USP Res:
7.9
0.04
USP Res:
7.9
0.02
0.02
0.00 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 Minutes 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60
0.00 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 Minutes 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60
ethinyl estradiol
Results: Rs = 1.1X Run time = 0.25X Pressure = 3.1X
levonorgestrel
2.1 x 75 mm Flow Rate: Pressure: Run Time:
XP, 2.5 μm 0.42 mL/min 4400 psi 2.00 min
0.06
2.1 x 75 mm Flow Rate: Pressure: Run Time:
XP, 2.5 μm 0.42 mL/min 4400 psi 2.00 min
Results: Rs = 1.1X Run time = 0.25X Pressure = 3.1X
AU
AU
0.06
2.1 x 50 mm UPLC, 1.7 μm Flow Rate: 0.61 mL/min Pressure: 7700 psi Run Time: 0.90 min.
levonorgestrel
Maximum benefits obtained on ACQUITY UPLC System
AU
0.04
USP Res:
7.0
ethinyl estradiol
0.02
Results: Rs = 1X Run time = 0.1X Pressure = 5.5X
0.00 0.00
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0.20
0.40
0.60
0.80 Minutes
1.00
1.20
1.40
1.60
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2014-3-24
Agenda
行业趋势制药行业应用
软饮料标准品测试
食品行业应用
柱筛选与方法开发应用注意事项
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软饮料测试
牛奶中的四环素（样品经SPE处理）
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Agenda
影响分离度的因素选择性最为重要
Rs =
N 4
行业趋势制药行业应用食品行业应用
α ?1 k α k +1
柱筛选与方法开发
应用注意事项
通过提高柱效改善分离: 较小的填料粒径较长的柱长柱装填良好柱硬件谱带展宽小[柱体积较小时] 系统死体积较小[柱体积较小时]
对分离度的影响
Double N Double k Double α %
通过以下手段改善分离: 提高保留选择性
改善程度
20 – 40% 15 – 20% > 400%
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影响选择性的主要因素
Ever-Growing Selectivity Range EverXBridge/BEH, XSelect CSH & HSS
ACQUITY HSS C18 SB XSelect HSS C18 SB
ACQUITY HSS PFP XSelect HSS PFP ACQUITY HSS CN XSelect HSS CN ACQUITY CSH Fluoro-Phenyl ACQUITY BEH Phenyl Xselect CSH Fluoro-Phenyl XBridge Phenyl ACQUITY BEH C8 XBridge C8 ACQUITY CSH Phenyl-Hexyl XSelect CSH Phenyl-Hexyl ACQUITY BEH Shield RP18 XBridge Shield RP18
ACQUITY HSS T3 XSelect HSS T3 ACQUITY BEH C18 XBridge C18 ACQUITY HSS C18 XSelect HSS C18 ACQUITY CSH C18 XSelect CSH C18
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可无忧转换的HPLC柱平台提供极佳的pH耐受性
eXtended Performance [XP] 2.5 μm Columns
可无忧转换至HPLC的 ACQUITY UPLC 颗粒技术平台
亚乙基桥杂化颗粒
130?, 200?, 300?
BEH
高强度硅胶颗粒
100?
HSS
表面带电杂化颗粒
130?
CSH
提供多种选择性
行业领先的化学稳定性
?
1.7 [UPLC], 2.5, 3.5, 5 and 10 μm
1.7 [UPLC], 2.5, 3.5 and 5 μm CSH 1.8 [UPLC], 2.5, 3.5 and 5 μm HSS
宽pH范围，耐受性最强 ? 通用性极佳 ? 固定相种类与柱规格丰富 ? 除通用于小分子化合物，还有专用于生物制药行业的BEH柱产品
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提高选择与保留
?T3
：增强对极性分子的反相保留能力 ?C18，C18SB：常规及特殊硅胶C18选择性 ?CN，PFP：特殊键合相
使选择性最大化
? ?
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独特的选择性低离子强度酸性条件下，对碱性化合物的高载量与优异峰形。 ?较宽pH范围，高低pH条件切换时平衡迅速 ?碱性分析物首选柱！
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选对柱选择性，快速建立方法示例：齐拉西酮碱降解物
选对柱选择性，保留分离高极性示例：生物胺（亲水作用色谱）
甲基-5-羟色胺羟色胺多巴胺肾上腺素去甲肾上腺素
N5-
C18
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选对柱性能，寿命更加长示例：盐酸利多卡因胶浆（含量）
Agenda
条件（按中国药典方法）流动相：磷酸盐缓冲溶液 pH8：乙腈 50:50 流速：1mL/min 色谱柱：XBridge 2.5um 4.6x75mm XP柱仪器：Alliance HPLC系统
LC
行业趋势制药行业应用食品行业应用柱筛选与方法开发
应用注意事项
- 柱外效应：柱外效应：柱规格与系统的适配性
- 检测频率的设置 - 流动相与滤膜
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系统扩散/谱带展宽影响柱效
系统对柱效的影响
Extra Column Within Column
谱带扩散: 1) 从进样器开始 2) 进入、进入、流经以及流出色谱柱 3) 进入检测器
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色谱柱与系统的匹配性
2.5 μm eXtended Performance Columns
Packed in ultra-low dispersion hardware
– Maintains XBridge or XSelect HPLC branding
Designed to withstand high pressure
– 4.6 mm ID capable of 9,000 PSI – 2.1 and 3.0 mm ID compatible with pressures UPLC
New 2.5 μm eXtended Performance XP Columns compatible with HPLC and UPLC Technology
Flexibility in configuration
– 2.1, 3.0 and 4.6 mm ID – 30, 50, 75，100 and 150mm lengths – 14 stationary phases
System
Particle Size Routine Pressure Column ID Column Length
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HPLC
3.5 μm, 5 μm < 4000 psi 4.6 mm ≤ 250 mm
UPLC
1.7/1.8 μm UPLC, XP 2.5 μm < 15000 psi (H-Class) 2.1 & 3.0 mm* ≤ 150 mm* 1.7/1.8 μm UPLC, < 18000 psi (I-Class) 1.0 & 2.1 mm* ≤ 150 mm*
XP 2.5 μm < 4000 psi 4.6 mm ≤ 75 mm
Packed with XBridge [BEH] and XSelect [CSH and HSS] 2.5 μm particles and chemistries
– BEH C18, Shield RP18, C8, Phenyl, HILIC and Amide – CSH C18, Phenyl-Hexyl and Fluoro-Phenyl – HSS C18, T3, C18 SB, Cyano and PFP
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(*) – additional UPLC-based bioseparation column configurations also available
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对采样频率设置的要求 1Hz vs. 20Hz （二氢苊测4.6x75mm柱效）
1. 防止流动相与吸滤头长菌
试管中的Milli-Q 纯水，取出培养，无微生物检出
已受污染的吸滤头，放在Milli-Q纯水中，再取水进行培养，有大量微生物检出。污染源！！
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因细菌污染所造成的峰形恶化
BEH200, SEC, 1.7um 4.6 x 150 mm 100% 水相流动相和纯水很容易长菌牢记: 每天新配水相流动相（或加入少量有机溶剂）牢记: 在关机前或系统长时间不用时，请先用水充分清洗系统，然后用10%适宜的有机溶剂（如乙腈或甲醇）清洗系统
2.
样品溶解性与样品制备：避免样品不溶微粒与样品沉积
过滤是必需的
– 总是使用0.2 μm 化学适宜的样品过滤膜使用保护柱也会有帮助离心对于去除微粒是必要的 – 通过离心可以去除所有的细颗粒,在4,000 到 12,000 rpm 下离心 5 到 30 分钟.
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多奈哌齐(Donepezil)片剂溶于稀释剂中，按USP方法制备，存在大量不溶性颗粒（非常糟糕）
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使用0.2μm滤膜过滤，仍存在非常细微的颗粒，溶液呈雾状 (仍然糟糕)
再加以离心步骤 12,000 rpm. 溶液完全澄清 (状态良好)
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3. 确保使用正确合格的过滤膜
案例：用户发现色谱柱寿命极短，经过排查，实际上色谱柱的入口端被乙腈溶解的膜材料主要是聚砜严重堵塞
色谱柱入口端被乙腈溶解的聚砜膜材料堵塞
4. 使用合格玻璃材质的溶剂瓶
案例：案例：色谱柱1: BEH UPLC 色谱柱在高pH (pH 10)条件下仅有100-200针的柱寿命色谱柱2: 新色谱柱前连接VanGuard? 保护柱，保护柱堵塞（保护了 UPLC色谱柱）
解决办法：更换滤膜
–
推荐使用：
o o
色谱柱出口端过滤片的内侧（靠近柱床一侧）被流动相溶解的硼硅玻璃严重堵塞解决办法：更换流动相瓶
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Waters P/N:186003524 0.2um 47mm GHP Waters P/N:WAT200531 0.2um 47mm PVDF
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解决办法: 使用Pyrex硼硅3.3的玻璃瓶
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Summary
行业趋势制药行业应用食品行业应用柱筛选与方法开发应用注意事项
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