HPLC设备的确认(中英)-Qualification-of-HPLC-Equipment.doc

PA/PH/OMCL (11) 04 - OMCL Guideline on qualification of HPLC equipment (Annex 1)

OMCL Network of the Council of Europe
QUALITY MANAGEMENT DOCUMENT
欧洲委员会OMCL网络
质量管理文件
PA/PH/OMCL (11) 04
QUALIFICATION OF EQUIPMENT
设备的确认
ANNEX 1: QUALIFICATION OF HPLC EQUIPMENT
附件1：HPLC (高效液相色谱仪) 设备的确认
Full document title and reference
文件全名和索引号
Qualification of Equipment 设备的确认
Annex 1: Qualification of HPLC equipment
PA/PH/OMCL (11) 04 附件1：HPLC设备的确认 PA/PH/OMCL (11) 04
Document type 文件类型
Guideline 指南
Legislative basis 立法基础
The present document was also accepted by EA as recommendation document to be used in the context of Quality Management System audits of OMCLs 本文件也被EA认可并推荐在OMCL的质量管理体系审计中使用。
Date of first adoption
首次采用日期
May 2005 2005年5月
Date of original entry into force
首次生效日期
June 2005 2005年6月
Date of entry into force of revised document 修订版生效日期
1st July 2011 2011年7月1号
Previous titles/other references
原文件名/其它索引号
This document replaces document PA/PH/OMCL (07) 17 DEF
本文件替代文件PA/PH/OMCL (07) 17 DEF
Custodian Organisation
监管组织
The present document was elaborated by the OMCL Network/ EDQM of the Council of Europe 本文件由OMCL网络/欧洲EDQM制定。
Concerned Network
相关网络
GEON

ANNEX 1 OF THE OMCL NETWORK GUIDELINE
“QUALIFICATION OF EQUIPMENT”

OMCL网络指南“设备的确认”之附件1
QUALIFICATION OF HPLC EQUIPMENT
HPLC设备的确认
Introduction 概述
The present document is the first Annex of the core document “Qualification of Equipment”, and it should be used in combination with it when planning, performing and documenting the HPLC equipment qualification process.
本文件是核心文件“设备的确认”第1个附件，在计划、实施和记录HPLC仪器的确认过程时，应将本文件与核心文件一起使用。

The core document contains the general introduction and the Level I and II of qualification, common to all type of instruments, and the present annex contains HPLC instrument-related recommendations on parameters to be checked and the corresponding typical acceptance Limits, as well as practical examples on the Methodology that can be used to carry out these checks.
核心文件包括了简介和第一阶段和第二阶段的确认，它们适用于所有类型的仪器。 本附件包括了HPLC仪器相关的需要检查的参数和相应典型的可接受标准的推荐，以及可用于进行这此检查的实用方法学举例。

When qualifying HPLC equipment, it should be noted that it is acceptable to check at Level III and IV several of the mentioned parameters at the same time in a combined test procedure (e.g. “overall” system performance test giving information on peak area precision, retention time precision, gradient reproducibility, etc).
在进行HPLC设备确认时，应注意也可以对第三阶段和第四阶段几个提到的参数采用联合检测程序同时进行检查（例如“全面”系统性能检查，同时给出峰面积精密度、保留时间精密度、梯度重复性等的信息）
TABLE III 表三
Level III. Periodic and motivated instrument checks
第三阶段 定期和专门的仪器检查
Examples of requirements for HPLC instruments and detectors
HPLC仪器和检测器要求举例
Instrument module
仪器模块
Parameter to be checked
需检查的参数
Typical tolerance Limits
典型误差限度
Solvent delivery system
溶剂输送系统
● Flow rate 流速
● Proportioning accuracy and precision (gradient test) 配比准确度和精密度（梯度测试）
● Proportioning ripple 配比波纹
●±5 %
●±2

●≤0.2 %
Injector
进样器
l Volume precision 进样量精密度
l Carry-over 残留
l RSD≤1.0 %

l see Annex I 见附件1
Autosampler
自动进样器
l Thermostatting accuracy and precision 恒温准确度和精密度
l ±3 °C
Oven or cooling device
柱温箱或冷却装置
l Thermostatting accuracy
恒温准确度
l ± 2 °C
UV/DAD detector
UV/DAD 检测器
l Linearity 线性
l Wavelength accuracy 波长准确度
l r2 ≥0.999
l ±2 nm
Fluorescence detector
荧光检测器
l Wavelength accuracy excitation波长准确度激发
l Wavelength accuracy emission 波长精密度发射
l Sensitivity 灵敏度
l ±3 nm

l ±3 nm

l ±see Annex I
Electrochemical detector 电化学检测器
l Accuracy of the signal 信号精密度
l Stability of the signal 信号稳定性
l see Annex I 见附件1

l see Annex I 见附件1
RID detector
RID 检测器
l Signal/Noise ratio 信噪比
l Drift over time 飘移
l see Annex I见附件1
l 0.1 mV/min
CD detector
CD 检测器
l Signal/Noise ratio 信噪比
l Sensitivity 灵敏度
l Drift over time 飘移
l Linearity 线性
l see Annex I见附件1
l see Annex I见附件1
l see Annex I见附件1
l r > 0.999

TABLE IV 表四
Level IV. In-use instrument checks
第四阶段 在用仪器的检查
Examples of requirements for HPLC instruments with UV or DAD detectors
配备UV或DAD检测器的HPLC仪器的要求举例
Parameter to be checked
需检查参数
Typical tolerance Limit 典型误差限度
l System suitability check for the Method 方法系统适用性检查
According to Ph. Eur. or MAH dossier or validated in-house Method
根据欧洲药典或MAH文件或公司内部经过验证的方法
l · Peak area precision (applicable to the main peaks in the test solution)
峰面积准确度（适用于供试液中主峰面积）
RSD ≤ 1.5 %
(unless otherwise prescribed in the system suitability of the Method, e.g. specific requirements from Ph. Eur. 2.2.46, API monographs or MA dossiers) 除非在方法的系统适用性里有描述，例如EP2.2.46特定要求，原料药各论或MA文件
l Retention time precision
保留时间精密度
RSD ± 5 %
l Carry-over (by comparing consecutive standard (of the substance being quantified) and blank injections) 残留（通过比较连续（进行定量检测的物质）标准和空白进样）
≤ 0.2 %
l Signal/Noise ratio (to be applied for related substances test)
信噪比（用于有关物质检测）
According to Ph. Eur.
根据欧洲药典

ANNEX I 附件1
Level III. Periodic and motivated instrument checks
第三阶段 定期和专门的仪器检查
This Annex contains practical examples of tests and their associated tolerance Limits for several parameters related to the performance of the different modules of a HPLC.
本附件包含的是检测HPLC仪器的不同模块性能的一些参数的实际例子及其参数的误差限度。

These examples can be considered by the OMCLs as possible approaches to perform the Level III of the equipment qualification process: “Periodic and motivated instrument checks”.
OMCLs 认为这些例子可以用来执行设备确认过程的阶段III“定期和专门的仪器检测”。

HPLC SOLVENT DELIVERY SYSTEM
HPLC 溶剂传输系统

The following tests are proposed for the periodic and motivated check of the HPLC solvent delivery system: flow rate and gradient test.
以下测试建议用于对HPLC溶剂传输系统的周期性主动检查：流速和梯度检测

FLOW RATE 流速

Materials: 材料
Volumetric flask of 5 or 10 ml
5ml或10ml的容量瓶
Calibrated chronometer
校正过的气压计

Settings: 设置
Mobile phase: degassed water
流动相：脱过气的水
No column (open end)*
无柱（开放式）*
Flow rate: adjusted between 0.5 and 3.0 ml/min
流速：调整到0.5至3.0ml/min之间
If high-pressure mixing systems are installed, this test has to be done on each solvent channel.
如果有安装高压混合系统，本测试应在每一溶剂通道进行。

* For certain equipment, e.g. in the case of low flow rates, the check would be performed by using a column or a backpressure regulator.
* 对于某些设备，例如，流速较低时，检测时需要用到一根色谱柱或背压调节器进行检查。

Method方法:
Set the flow rate at an appropriate level and measure the time needed to fill the volumetric flask up to the mark. Record the time needed.
将流速设定在一个合适的水平，测量将容量瓶装满至刻度的时间。记录所用的时间。
  f ＝V /t   f ＝(V×60)/t
f ................. measured flow rate [ml/min] 测得的流速
t ................. elapsed time to fill up to mark [s] 充满至刻度所需的时间
V ............... volume of the volumetric flask [ml] 容量瓶的体积
  D＝100×(f－F)/F
D ............... deviation [%] 偏差
F ............... adjusted flow rate [ml/min] 调节后的流速
f ................. measured flow rate [ml/min] 测量的流速
Limits限度: ±5%

GRADIENT COMPOSITION AND RIPPLE 梯度组成和波动

Settings: 设置
Stainless steel capillary e.g. 2000 x 0.12 mm installed instead of a column
不锈钢毛细管，例如2000X0.12mm，代替色谱柱安装于仪器
Detection: UV-Detector adjusted to 265 nm
检测器：紫外检测器调节至265nm
Mobile phase A: degassed water
流动相A：脱气水
Mobile phase B: degassed water containing 0.5% acetone
流动相B：含0.5%的脱气水
Flow rate: 1.0 ml/min
流速：1.0ml/分钟

Method方法:
The test is carried out in the following way by using a gradient program depending on the number of solvent channels and the configuration of the system:
根据溶剂通道的数量和系统的配置，使用下列不同系统参数和梯度进行测试：
A-B
A-B and A-C
A-C, A-B and B-D
time [min]
时间【分钟】
% mobile phase A (water)
%流动相A（水）
% mobile phase B
% 流动相B
(water-acetone mixture)
（水-丙酮混合物）
0.0
100
0
0.1
90
10
10
90
10
10.1
50
50
20
50
50
20.1
10
90
30
10
90
30.1
0
100
40
0
100
40.1
100
0
Start the test by pumping water for at least 10 min to equilibrate the system.
开始测试前，先用水平衡系统至少10分钟。
The zero % value at the start of the test is the baseline. All steps are measured at the beginning of the horizontal part of the line either by software or manually on the paper print using a liner. The height of the 100% water/acetone mixture is used as the 100% value in the following calculation.
测试开始时的0%值是基线值。所有步骤均在基线的水平部分开始时由软件或人工测量，并打印在纸上。100%水/丙酮混合物的高度在以下计算中作为100%值。

  %H＝100* h/H
%H ............ calculated composition 计算组成
h ................ height of the measured line 测量线的高度
H ............... height of the 100% water/acetone mixture line (mobile phase B)
100%水/丙酮混合物线的高度（流动相B）
  d ＝%H－G
d ................ deviation 偏差
G ............... gradient composition adjusted [% acetone/water solution mixture = mobile phase B]
梯度组成调节【%丙酮/水溶液混合物 = 流动相B】

Limits限度:
Absolute deviation: ±2 of the adjusted value
绝对偏差： 调整值的±2
The ripple of the gradient composition is the percentage of noise of the 50% line from the gradient program.
梯度组分的波动为梯度程序噪声基线的50%

  %R ＝100* N /h50
%R ............ ripple 波纹
h50 ............. height of the 50% line 50%线的高度
N ............... height of the noise line, measured during 1 minute in the linear region 噪声线高度，在线性区域1分钟期间测量的结果

Limits 限度: ≤ 0.2 %

HPLC INJECTOR
HPLC进样器

Volume precision and carry-over are the tests proposed for the periodic and motivated check of the HPLC injector.
建议在HPLC进样器的定期和专门检查中进行容量(体积)精密度和残留检查。

VOLUME PRECISION AND CARRY-OVER
体积精密度和残留
Solutions: 溶液
Solvent A: methanol : water R, 60 : 40.
溶液 A：乙醇：水（试剂） = 60：40
Reference solution (a): dissolve 15.0 mg methyl-, ethyl-, and propylparabene in 100.0 ml of solvent A.
对照液（a）：取15.0mg尼泊金M、尼泊金乙酯和尼泊金丙酯溶于100.0ml的溶剂A中
Reference solution (b): Dilute 1.0 ml of reference solution (a) to 10.0 ml of solvent A.
对照液（b）：取1.0ml对照液（a）用溶于10.0ml溶剂A中
Reference solution (c): Dilute 1.0 ml of reference solution (b) to 100.0 ml of solvent A.
对照液（c）：取1.0ml对照液（b）用溶于100.0ml溶剂A中

Settings: 设置
Column: Lichrospher 100 RP8, 5um, 125 x 4 mm, without precolumn
色谱柱：Lichrospher 100 RP8, 5 um, 125 x 4 mm, 无衬管
Mobile phase: methanol : water = 60 : 40
流动相：乙醇：水 = 60：40
Flow rate: 1.0 ml/min
流速：1.0ml/分钟
Detection: 254 nm
检测器：254nm
Injection volume: 20 μl
进样体积：20ul

Method方法:
Injection scheme: 进样序列
— 6x reference solution (b) 对照液（b）6针
— 1x reference solution (a) 对照液（a）1针
— 1x solvent A (blank injection 1) 溶剂A（空白1）1针
— 1x reference solution (b) 对照液（b）1针
— 1x solvent A (blank injection 2) 溶剂A（空白2）1针
— 1x reference solution (c) 对照液（c）1针

Limit限度s:
Repeatability of peak areas: The relative standard deviation of the peak areas of all peaks in the chromatogram obtained with the reference solution (b) should be ≤1.0 %.
峰面积重复性：对照液（b）色谱图中所有峰的峰面积相对标准偏差应≤1.0 %

Carry-over: The percentage of the peak area corresponding to propylparabene in the blank injection 1 does not exceed 0.5% of 10 times the peak area of the propylparabene peak in the chromatogram obtained with the reference solution (b) injected after the blank injection.
残留：尼泊金丙酯在空白图谱中的峰面积不超过在空白后进样的对照液（b）中尼泊金丙酯的对应峰面积的10倍的0.5%

The percentage of the peak area corresponding to propylparabene in the reference solution (c) is 0.9 – 1.1% of the peak area of the propylparabene peak in the chromatogram obtained with the reference solution (b) injected after the blank injection.
在对照液（c）中尼泊金丙酯对应的峰面积为空白后进样的对照液（b）中尼泊金丙酯峰面积的0.9-1.1%。

HPLC AUTOSAMPLER
HPLC 自动进样器

Thermostatting accuracy and precision can be tested in the frame of the periodic and motivated check of the HPLC Autosampler.
对HPLC自动进样器的定期和专门检查可以检测温度的准确度和精确度。

THERMOSTATTING ACCURACY 温度准确度
Materials: 材料
Calibrated temperature probe.
校正过的温度探头

Method方法:
Select a temperature along the operational or required temperature range of the equipment. Wait until the system is equilibrated.
在可操作范围或要求的温度范围内选择并设定一个温度，等待系统平衡
By means of the calibrated probe, measure the actual temperature in the autosampler and compare it to the selected temperature.
采用校正过的温度探头，测量自动进样器的实际温度，与设定的温度进行比较
Repeat the same procedure at different pre-selected points covering the temperature range.
在选定的温度范围内的不同温度点重复相同的步骤

Limit限度s:
The actual temperature may not differ more than ±3°C with respect to the selected temperature.
实际温度与设定的温度相比差值不超过±3°C

THERMOSTATTING PRECISION 温度精确度

Materials: 材料
Calibrated temperature probe.
校正过的温度探头。

Method方法:
Select a temperature along the operational or required temperature range of the equipment. Wait until the system is equilibrated.
选择一个操作温度或要求的仪器温度范围，等待系统平衡。
By means of the calibrated probe, make “n” measurements over a pre-established period of time. Compare the mean of the “n” measurements to the selected temperature.
采用校正过的温度探头，在预设的时间内进行n次测量，比较n次测量值的平均值与设定的温度。

Limits限度:
The actual temperature may not differ more than ±3°C with respect to the selected temperature.
实际温度与设定的温度之间差值不超过±3°C

HPLC OVEN/COOLING DEVICE 色谱仪柱温箱/降温设施

Thermostatting accuracy is the parameter tested in this example of periodic and motivated check of the HPLC oven/cooling device.
温度的准确度是对HPLC柱温箱/降温设施进行定期和专门检查时检测的参数。

THERMOSTATTING ACCURACY 温度准确性

Materials: 材料
Calibrated thermometer.
校正过的温度计

Method方法:
Set the column oven temperature to 40 °C, wait about 30 minutes to equilibrate the system, put a calibrated thermometer into the oven and read the temperature after 10 minutes.
设定柱温箱温度为40 °C，等待约30分钟平衡系统，将校正过的温度计放入柱温箱中，在10分钟后读取温度。

Limits限度: 38 - 42°C.

HPLC UV/DAD DETECTOR
HPLC 紫外/DAD检测器
The periodic and motivated check of the HPLC UV/DAD detector can be performed by testing the linearity and the wavelength accuracy.
对HPLC紫外/DAD检测器的定期和专门的检查，可以对线性和波长准确性进行检测。

LINEARITY 线性

Solutions: 溶剂配制
Std. 1: 0.5 μg caffeine/1 ml methanol HPLC Grade
标准1： 0.5 μg咖啡因/1ml色谱级甲醇
Std. 2: 1.0 μg caffeine/1 ml methanol HPLC Grade
标准2： 1.0 μg咖啡因/1ml色谱级甲醇
Std. 3: 5.0 μg caffeine/1 ml methanol HPLC Grade
标准3： 5.0 μg咖啡因/1ml色谱级甲醇
Std. 4: 25.0 μg caffeine/1 ml methanol HPLC Grade
标准4：25.0 μg咖啡因/1ml色谱级甲醇
Std. 5: 50.0 μg caffeine/1 ml methanol HPLC Grade
标准5：50.0 μg咖啡因/1ml色谱级甲醇
Std. 6: methanol HPLC Grade (blank)
标准6：色谱级甲醇（空白）

Std. 5: weigh 9.0 to 11.0 mg caffeine and fill up to 200.0 ml with methanol HPLC
标准5：称取9.0至11.0mg咖啡因，用HPLC级别甲醇稀释至200.0ml
Std. 4: dilute 50.0 ml of Std. 5 to 100.0 ml with methanol
标准4：取50.0ml标准5溶液用甲醇稀释至100.0ml
Std. 3: dilute 10.0 ml of Std. 5 to 100.0 ml with methanol
标准3：取10.0ml标准溶液5用甲醇稀释至100.0ml
Std. 2: dilute 20.0 ml of Std. 3 to 100.0 ml with methanol
标准2：取20.0ml标准3溶液用甲醇稀释至100.0ml
Std. 1: dilute 10.0 ml of Std. 3 to 100.0 ml with methanol
标准1：取10.0ml标准3用甲醇稀释至100.0ml

Settings: 设置
Column: RP-18 5 um 30-50 x 2,1-4.6 mm or capillary 2000 mm x 0.12 mm ID
色谱柱：RP-185 um 30-50 x 2,1-4.6 mm or 毛细管柱 2000 mm x 0.12 mm ID
Mobile phase: methanol HPLC Grade
流动相：色谱级甲醇
Oven temperature: 40 °C
柱温箱温度：40 °C
Flow rate: 1.0 ml/min (adjusted by using 100% methanol)
流速：1.0ml/min（用100%甲醇调整）
Detection: 273 nm
检测波长：273nm
Injection volume: 20μl
进样量：20ul

Method方法:
Injection scheme:
进样序列
2