HPLC设备的确认(中英) Qualification of HPLC Equipment解读

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 Guideline 指南 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。 May 2005 2005年5月 June 2005 2005年6月 1st July 2011 2011年7月1号 This document replaces document PA/PH/OMCL (07) 17 DEF 本文件替代文件PA/PH/OMCL (07) 17 DEF Document type 文件类型 Legislative basis 立法基础 Date of first adoption 首次采用日期 Date of original entry into force 首次生效日期 Date of entry into force of revised document 修订版生效日期 Previous titles/other references 原文件名/其它索引号 1st July 2011 Page 1 of 18

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

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。 GEON Concerned Network 相关网络

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）

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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 Parameter to be checked Typical tolerance Limits 仪器模块 需检查的参数 典型误差限度 Solvent delivery system ● Flow rate 流速 ●±5 % ● Proportioning accuracy and 溶剂输送系统 ●±2 precision (gradient test) 配比准 确度和精密度（梯度测试） ●≤0.2 % ● Proportioning ripple 配比波纹 Injector  Volume precision 进样量精密 RSD≤1.0 % 进样器 度  Carry-over 残留  see Annex I 见附件1 Autosampler  Thermostatting accuracy and  ±3 °C 自动进样器 precision 恒温准确度和精密度 Oven or cooling device  Thermostatting accuracy  ± 2 °C 柱温箱或冷却装置 恒温准确度 UV/DAD detector  Linearity 线性  r2 ≥0.999 UV/DAD 检测器  Wavelength accuracy 波长准 ±2 nm 确度 Fluorescence detector  Wavelength accuracy excitation ±3 nm 荧光检测器 波长准确度激发  Wavelength accuracy emission  ±3 nm 波长精密度发射  Sensitivity 灵敏度  ±see Annex I Electrochemical  Accuracy of the signal 信号精 see Annex I 见附件1 detector 电化学检测密度 器  Stability of the signal 信号稳定 see Annex I 见附件1 性 RID detector  Signal/Noise ratio 信噪比  see Annex I见附件1  0.1 mV/min RID 检测器  Drift over time 飘移 CD detector  Signal/Noise ratio 信噪比  see Annex I见附件1 CD 检测器  Sensitivity 灵敏度  see Annex I见附件1  Drift over time 飘移  see Annex I见附件1  r > 0.999  Linearity 线性

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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 Typical tolerance Limit 典型误差限度 Parameter to be checked 需检查参数  System suitability check for the According to Ph. Eur. or MAH dossier or validated in-house Method Method 方法系统适用性检查 根据欧洲药典或MAH文件或公司内部经过验证的方法  RSD ≤ 1.5 % the main peaks in the test solution) (unless otherwise prescribed in the system 峰面积准确度（适用于供试液中主峰suitability of the Method, e.g. specific requirements from Ph. Eur. 2.2.46, API 面积） 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  Retention time precision RSD ± 5 % 保留时间精密度  Carry-over (by comparing ≤ 0.2 % 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）  Signal/Noise ratio (to be applied for According to Ph. Eur. related substances test) 根据欧洲药典 信噪比（用于有关物质检测）

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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。 1st July 2011 Page 5 of 18

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＝ ＝(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 % mobile phase B time [min] % mobile phase A (water) % 流动相B (water-acetone mixture) 时间【分钟】 %流动相A（水） （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 1st July 2011 Page 6 of 18

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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。

＝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） ＝%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%

＝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

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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: 2 nm 检测器：2nm

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 1st July 2011 Page 8 of 18

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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。

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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)

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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 x blank 2针空白 1 x Std. 1 1 x Std. 2 1 x Std. 3 1 x Std. 4 1 x Std. 5

标准1-5依次各一针

Limits限度: r2 ≥ 0.999

Remark: As this test employs different test solutions to be injected, it covers also the check of correct positioning vials in the autosampler.

注：由于此检测中要进样不同供试溶液，因此也包括了对自动进样器样品瓶位置正确性的检查。

WAVELENGTH ACCURACY 波长准确度

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If there are built-in test procedures for the determination and adjustment of wavelength accuracy, follow the instructions of the instrument manual.

如果内置检测程序可以检测和调节波长准确度，则根据操作手册进行操作 In all other cases use the procedure described below. 否则，就采用以下程序

Solutions: 溶液

DAD: caffeine Std. 5 from the linearity testing 线性测试中的咖啡因5号标准溶液 UV/VIS: 1.0 g/ml anthracene solution in water 1.0 g/ml的蒽水溶液

Settings: 设置

Mobile phase: 15% acetonitrile in water 流动相：15%乙腈水溶液

-50 x 2,1-4.6 mm or capillary 2.0 m x 0.12 mm ID

色谱柱：RP18，5μm, 30-50×2.1-4.6mm或毛细管柱 20m×0.12mm内径 Oven temperature: 40 °C 柱箱温度：40℃

Flow rate: 1.0 ml/min (adjusted by using 15% acetonitrile in water) 流速：1.0ml/min（用15%的乙腈水溶液调节） Detection: scan from 230 nm to 290 nm (DAD) 检测：自230至290nm扫描（DAD） Injection volume: 20 μl 进样体积：20μl

Method方法:

DAD: Inject 20μl of the caffeine solution and record the spectrum. The maximum is at 272 nm and the minimum at 244 nm.

DAD: 咖啡因溶液进样20μl，记录色谱图，最大吸收波长为272nm，最小为244nm.

UV/VIS: fill the cell with the anthracene solution and change the wavelength from 248 to 2 nm in 1 nm steps; record the maximum of absorption. The theoretical value is 251 nm.

UV/可见光：向池中注入蒽溶液，将波长以1nm的步距从248改为2nm，记录最大吸收值。理论值为251nm。

Limits限度: ≤2 nm

HPLC FLUORESCENCE DETECTOR HPLC 荧光检测器

The following three parameters are proposed for the performance of the periodic and motivated check of the HPLC fluorescent detector:

下面三个参数建议作为对荧光检测器性能进行定期和专门检查时的参数。

WAVELENGTH ACCURACY EXCITATION 波长激发准确度

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Method方法:

Rinse and fill the measuring cell with de-ionized water 用去离子水淋洗并充满检测池

Adjust the excitation wavelength to 350 nm. 调节激发波长为350nm

Measure the emission and subtract 397 nm (theoretical value). 测量发射波长并减去397nm（理论值）

Limits 限度: ±3 nm

WAVELENGTH ACCURACY EMISSION 波长发射准确度

Method方法: 方法

Rinse and fill the measuring cell with de-ionized water 用去离子水淋洗并充满检测池

Adjust the emission wavelength to 397 nm. 调节发射波长为397nm

Measure the excitation and subtract 350 nm (theoretical value). 测量激发波长并减去350nm（理论值）

Limits 限度:±3nm

SENSITIVITY 灵敏度

Solutions: 溶液

Quinine HCl.2H2O solution conc. 0.015μg/ml (=15 ppb)

二水盐酸喹啉溶液，浓度0.015μg/ml (=15 ppb)

The quinine solution is prepared with the following mobile phase: dissolve 6.8 g of potassium dihydrogen

phosphate R and 3.0 g of hexylamine R in 700 ml of water R, adjust to pH 2.8 with dilute phosphoric acid R, add 90 ml of acetonitrile R and dilute to 1000.0 ml with water R.

喹啉溶液用以下流动相制备：溶解6.8g磷酸二氢钾和3.0g己胺于700ml试剂级水中，用稀磷酸调pH至2.8，加90ml乙腈并用试剂级水稀释至1000.0ml.

Settings:设置

The chromatographic conditions are set according to Ph. Eur. “Quinine HCl” (01/2005:0018), test “Other cinchona alkaloids”, with modified flow rate and acetonitrile concentration. 色谱条件设定依据欧洲药典“盐酸喹啉”（01/2005:0018），检测“其他金鸡纳生物碱”，用不同的流速和乙腈浓度。

Mobile phase: as above 流动相：同上

Column: RP18, 5 μm, 250 × 4.6 mm 色谱柱: RP18, 5 μm, 250 ×4.6 mm Flow rate: 1.2 ml/min

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流速：1.2 ml/min

Excitation wavelength: 350 nm 激发波长：350nm

Emission wavelength: 397 nm 发射波长：397nm

Flow-cell volume: 8 μl (for this example, a Waters 2475 Multi Fluorescentie detector was used. Flow-cell volume may vary depending on the instrument manufacturer)

流动池体积：8μl（此例中，使用的是Waters2475多荧光检测器，流动池体积可能因不同的仪器厂商而不同）

Method方法:

Inject 10μl of the quinine solution and measure the peak height. 进样喹啉溶液10μl，测量峰高度

Inject 10μl of the blank and measure the peak height of the noise. 进样空白溶液10μl，测量噪音峰高

Divide the peak height of the quinine solution by 3 times the peak height of the noise. 将喹啉溶液峰高除以3倍噪音峰高

Divide the concentration of the quinine solution by the previously obtained factor. 将喹啉溶液浓度除以之前的系数 Limit限度: ≤ 0.5 ppb

HPLC ELECTROCHEMICAL DETECTOR HPLC 电化学检测器

Accuracy and stability of the signal are the proposed parameters to be tested during the periodic and motivated check of the HPLC electrochemical detector.

电化学检测器的信号准确度和稳定性是对其定期和专门检测中建议检测的参数。

ACCURACY AND STABILITY OF THE SIGNAL 信号准确度和稳定性

Settings: 设定

Cell potential of a dummy cell: 800 mV 空白池电压：800mv Rise time filter: 0.1 s 增加时间过滤：0.1s Range: 0.1 nA 范围：0.1nA Temperature: 30 °C 温度：30℃

Method方法:

Accuracy: Measure the electric current and subtract 2.67 nA (theoretical value) 准确度：检测电流并减去2.67nA（理论值）

Stability: Measure the noise over a period of 5 minutes

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稳定性：连续5分钟测量噪音

Limits 限度:

准确度（池电流）：0.05nA

Stability of the signal (Noise): max. 2 pA or 20 mV 信号（噪音）稳定性：最大2pA或20mV

HPLC RID DETECTOR

HPLC的 RID (折射率) 检测器

Signal to Noise ratio and drift over time are the parameters proposed for the periodic and motivated check of the HPLC RID (refractive index) detector.

HPLC的RID（折射率）检测器的信噪比和时间漂移是定期和专门检测中建议检测的参数

SIGNAL TO NOISE RATIO 信噪比

Solutions溶液:

Standard solution: D-fructose concentrate solution at 4.0 mg/ml (dilute 200.0 mg fructose + 20 ml water + 25.0 ml acetonitrile up to 50.0 ml with water for HPLC)

标准溶液：D-左旋糖溶液4.0mg/ml（将200.0mg左旋糖+20ml水+25.0ml乙腈，用色谱纯水稀释至50.0ml）

Settings设置:

Column: spherisorb NH2 (or equivalent) 250 ×4.6 mm or other 色谱柱：spherisorb NH2 (或相当的) 250 ×4.6 mm 或其他 Oven temperature: 38°C 柱温箱温度：38℃ Flow rate: 1.0 ml/min 流速: 1.0 ml/min

Injection volume: 20 μl 进样量: 20 μl

Mobile phase: 0.253g sodium hydrogen phosphate R in 220 ml + 780 ml acetonitrile 流动相：0.253g磷酸氢二钠溶于220ml+780ml乙腈

Method方法:

After equilibration, inject three times a blank solution of mobile phase over a run time where the system is stable. Measure the baseline noise over an appropriate period.

平衡后，以流动相为空白进样三针，系统稳定运行适当时间，测定适当时长内噪音基线 The baseline noise is accepted if the mean height of the three replicates is < 1000 μV. 可接受基线噪音：三次平行进样平均值< 1000 μV

To calculate the signal to noise ratio, inject three times a solution of fructose at 0.4 mg/ml and calculate the mean of the three replicates.

计算信噪比，左旋糖溶液0.4mg/ml进样三针，计算平均值

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Limit限度: S/N ≥10 限度：信噪比≥10

DRIFT OVER A DEFINED PERIOD OF TIME 指定时长的漂移

Method方法:

Calculate the slope of the amplitude of random variations in the detector’s signal over 1 minute. 计算检测器信号在1分钟内随机波动幅度的斜率

Limits 限度: ±0.1 mV/min

Alternatively, the requirement may be expressed in △RI/min or in % of full scale of the selected range.

或者，也可以表述为△RI/min或者所选量程的全范围的%。

HPLC CD DETECTOR HPLC的CD检测器

The following tests are proposed to perform the periodic and motivated check of the HPLC CD (circular dichroism) detector.

对HPLC的CD (圆二图谱) 检测器进行定期和专门检查时，建议进行以下检测。

LINEARITY AND SIGNAL TO NOISE RATIO 线性和信噪比

Solutions: 溶液

Reference solution (a): dissolve 25.0 mg D(-) pantolactone in 50.0 ml water 对照溶液（a）：溶解25.0mg D(-)泛酰内酯于50.0ml水中

Reference solution (b): dilute 2.0 ml of reference solution (a) to 10.0 ml with water 对照溶液（b）：取对照溶液（a）2.0ml用水稀释至10.0ml

Reference solution (c): dilute 4.0 ml of reference solution (a) to 10.0 ml with water 对照溶液（c）：取对照溶液（a）4.0ml用水稀释至10.0ml.

Reference solution (d): dilute 6.0 ml of reference solution (a) to 10.0 ml with water 对照溶液（d）：取对照溶液（a）6.0ml用水稀释至10.0ml.

Reference solution (e): dilute 8.0 ml of reference solution (a) to 10.0 ml with water 对照溶液（e）：取对照溶液（a）8.0ml用水稀释至10.0ml.

Reference solution (f): dilute 0.5 ml of reference solution (b) to 25.0 ml with water 对照溶液（f）：取对照溶液（b）0.5ml用水稀释至25.0ml.

Settings: 设定

Column 色谱柱: C18, 150 ×4 mm, 5 μm

Mobile phase 流动相: acetonitrile乙腈 : water水 = 10 : 90 Flow 流速: 1.0 ml/min

Detection 检测波长: 225 nm Injection volume 进样量: 20 μl

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Method方法:

Check the linearity of de CD- and UV-signal of D(-)pantolactone reference solution a,b,c,d,e. 检查CD和UV信号与D(-)泛酰内酯对照溶液a,b,c,d,e的线性关系

Measure the noise of the CD-signal of reference solution (f) between 0 – 10 min. 检查0-10分钟内对照溶液(f)的CD信号的噪声

- Calculate the absolute concentration (μg) in the cell

计算池中绝对浓度(μg)

- Calculate the signal-to-noise ratio (S/N) for 0.01 μg in the cell 计算池中0.01 μg信噪比（S/N）

- Calculate the sensitivity with the calculated S/N and the specified S/N= 2 (0.01 x 2/ S/N

calculated)

- 计算所得信噪比及信噪比为2(0.01 x 2/ S/N calculated)时的灵敏度

Limits 限度:

Linearity: The linearity of the calibration line obtained with reference solution (a,b,c,d,e) should be r > 0.999.

线性：由对照液（a,b,c,d,e）所得的校正曲线的线性应是r> 0.999。 Sensitivity: The sensitivity at S/N= 2 should be better than 0.01μg. 灵敏度：信噪比为2时灵敏度应好于0.01μg。

S/N ratio: The Limit for S/N is > 1.0 and the sensitivity should be 0.020 μg at maximum. 信噪比：信噪比的限度为＞1.0时，灵敏度最大值为0.020μg。

DRIFT OVER A DEFINED PERIOD OF TIME 指定时长的飘移

Settings设定:

Column 色谱柱: C18, 150 x 4 mm, 5μm

Mobile phase 流动相: acetonitrile乙腈 : water水 = 10 : 90 Flow 流速: 1.0 ml/min

Detection 检测波长: 290 nm Injection volume 进样量: 20μl

Method方法:

Inject water and stop the flow after 5 minutes. Measure the CD-signal for 1 hour. 进水样，5分钟后停止流动。测量CD信号，持续1小时。

Measure with the cursor the drift of the baseline between 5 and 65 min. 用指针测量5到65分钟之间的基线飘移

Limits限度:

Not more than 0.1 mdeg/h 不超过0.1 mdeg/h

SPECTRA COMPARISON 谱图比较

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Solutions 溶液:

Reference solution (a): dissolve 5.0 mg dexamethasone in 10.0 ml 40% acetonitrile 对照溶液（a）：溶解5.0mg 地塞米松于10.0ml 40%乙腈中。

Settings设定:

Column 色谱柱: C18, 150 x 4 mm, 5μm

Mobile phase 流动相: acetonitrile乙腈 : water水 = 40 : 60 Flow 流速: 1.0 ml/min

Detection 检测波长: 230 nm Injection volume 进样量: 20ul

Method方法:

Compare the maxima/minima obtained at the Installation of the detector (see table). 比较检测器安装结果（见下表） CD max 最大 CD min 最小 UV max 最大 222 nm 224 nm 236 nm 230 nm 252 nm 284 nm Limits限度:

The maxima and minima may not differ more than± 4 nm. 最大和最小值之差不可超过4nm。

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