儀器確認(rèn)附件1HPLC確認(rèn)中英文.doc

1、pa/ph/omcl (11) 04 - omcl guideline on qualification of hplc equipment (annex 1) omcl network of the council of europe quality management document pa/ph/omcl (11) 04qualification of equipment儀器確認(rèn)annex 1: qualification of hplc equipment附件1：hplc儀器的確認(rèn)full document title and reference 文件全名和索引號qualificat

2、ion of equipment annex 1: qualification of hplc equipment pa/ph/omcl (11) 04 document type 文件類型guideline 指南legislative basis 立法基礎(chǔ)the present document was also accepted by ea as recommendation document to be used in the context of quality management system audits of omcls date of first adoption 首次發(fā)行日

3、期may 2005 date of original entry into force 首次執(zhí)行日期june 2005 date of entry into force of revised document 修訂后執(zhí)行日期1st july 2011 previous titles/other references 原文件名/其它索引號this document replaces document pa/ph/omcl (07) 17 def custodian organisation 保管機(jī)構(gòu)the present document was elaborated by the omcl n

4、etwork/ edqm of the council of europe concerned network 相關(guān)網(wǎng)絡(luò)geon annex 1 of the omcl network guideline“qualification of equipment”omcl網(wǎng)絡(luò)指南“儀器的確認(rèn)”之附件1qualification of hplc equipmenthplc儀器的確認(rèn)introduction 概述the present document is the first annex of the core document “qualification of equipment”, and i

5、t 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“儀器的確認(rèn)”第1個附件，在計劃、實(shí)施和記錄hplc儀器的確認(rèn)過程時，應(yīng)將本文件與核心文件一起使用。the core document contains the general introduction and the level i and ii of qualification, common to all type of ins

6、truments, 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. 核心文件包括了第一級和第二級確認(rèn)的通用介紹，適用于所有類型的儀器，本附件包括了hplc儀器相關(guān)的需

7、要檢查的參數(shù)和相應(yīng)典型的可接受標(biāo)準(zhǔn)的推薦，以及可用于進(jìn)行這此檢查的實(shí)用方法學(xué)舉例。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 pre

8、cision, retention time precision, gradient reproducibility, etc).在進(jìn)行hplc儀器確認(rèn)時，應(yīng)注意也可以對第三級和第四級幾個提到的參數(shù)采用聯(lián)合檢測程序同時進(jìn)行檢查（例如“全面”系統(tǒng)性能檢查，同時給出峰面積精密度、保留時間精密度、梯度重復(fù)性等的信息）table iii 表三level iii. periodic and motivated instrument checks第三級 定期主動儀器檢查examples of requirements for hplc instruments and detectorshplc儀器和檢測器

9、要求舉例instrument module 儀器模塊parameter to be checked 需檢查的參數(shù)typical tolerance limits典型允差限度 solvent delivery system溶劑傳送系統(tǒng) flow rate 流速 proportioning accuracy and precision (gradient test) 配比準(zhǔn)確度和精密度（梯度測試） proportioning ripple 配比波紋±5 % ±2 0.2 % injector 進(jìn)樣器l volume precision 進(jìn)樣量精密度l carry-over 殘留

10、l rsd1.0 % l see annex i 見附件1autosampler 自動進(jìn)樣器l thermostatting accuracy and precision 恒溫準(zhǔn)確度和精密度l ±3 °c oven or cooling device柱溫箱或冷卻裝置 l thermostatting accuracy 恒溫準(zhǔn)確度l ± 2 °c uv/dad detector uv/dad檢測器l linearity 線性l wavelength accuracy 波長準(zhǔn)確度l r2 0.999 l ±2 nm fluorescence det

11、ector 熒光檢測器l wavelength accuracy excitation波長準(zhǔn)確度激發(fā) l wavelength accuracy emission 波長精密度發(fā)射l sensitivity 靈敏度l ±3 nm l ±3 nm l ±see annex i electrochemical detector 電化學(xué)檢測器l accuracy of the signal 信號精密度l stability of the signal 信號穩(wěn)定性l see annex i 見附件1l see annex i 見附件1rid detector rid檢測器l

12、 signal/noise ratio 信噪比l drift over time 飄移l see annex i見附件1l 0.1 mv/min cd detector cd檢測器l signal/noise ratio 信噪比l sensitivity 靈敏度l drift over time 飄移l linearity 線性l see annex i見附件1l see annex i見附件1l see annex i見附件1l r > 0.999 table iv 表四level iv. in-use instrument checks第四級 在用儀器檢查examples of re

13、quirements for hplc instruments with uv or dad detectors配備uv或dad檢測器的hplc儀器的要求舉例parameter to be checked 需檢查參數(shù)typical tolerance limit限度s l system suitability check for the method方法系統(tǒng)適用性檢查 according to ph. eur. or mah dossier or validated in-house method根據(jù)歐洲藥典或mah文件或公司內(nèi)部經(jīng)過驗證的方法l · peak area precis

14、ion (applicable to the main peaks in the test solution) 峰面積準(zhǔn)確度（適用于供試液中主峰面積）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) 除非在方法的系統(tǒng)適用性里有描述，例如ep2.2.46特定要求，原料藥各論或ma文件l retention time precisio

15、n 保留時間精密度rsd ± 5 %l carry-over (by comparing consecutive standard (of the substance being quantified) and blank injections) 殘留（通過比較連續(xù)（進(jìn)行定量檢測的物質(zhì)）標(biāo)準(zhǔn)和空白進(jìn)樣） 0.2 % l signal/noise ratio (to be applied for related substances test) 信噪比（用于有關(guān)物質(zhì)檢測）according to ph. eur. 根據(jù)歐洲藥典annex i 附件1level iii. periodic

16、 and motivated instrument checks第三級 定期性的主動儀器檢查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. 本附件包括一些實(shí)用幾個與hplc不同模塊相關(guān)的參數(shù)檢測舉例及其相應(yīng)的可接受限度。these examples can be considered by the om

17、cls as possible approaches to perform the level iii of the equipment qualification process: “periodic and motivated instrument checks”. 這些例子可以認(rèn)為是omcl實(shí)驗室可能的進(jìn)行第三級儀器確認(rèn)的方法“周期主動儀器檢查”hplc solvent delivery system 高壓液相溶劑傳輸系統(tǒng)the following tests are proposed for the periodic and motivated check of the hplc so

18、lvent delivery system: flow rate and gradient test. 以下測試作為hplc溶劑傳輸系統(tǒng)周期性主動檢查：流速和梯度檢查flow rate 流速materials: 材料volumetric flask of 5 or 10 ml 5ml或10ml的容量瓶calibrated chronometer 校正過的氣壓計settings: 設(shè)置mobile phase: degassed water 流動相：脫過氣的水no column (open end)* 無柱（開放式）*flow rate: adjusted between 0.5 and 3.0

19、 ml/min 流速：調(diào)節(jié)在0.5到3.0ml/minif high-pressure mixing systems are installed, this test has to be done on each solvent channel. 如果有安裝高壓混合系統(tǒng)，本測試應(yīng)在每一溶劑通道進(jìn)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. * 對于一定的儀器，例如，

20、流速較低時，采用一根柱子或背壓調(diào)整器進(jìn)行檢查 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. 將流速設(shè)定在一個合適的水平，測量將容量瓶裝滿至刻度的時間。記錄所用的時間。  f v /t   f (v×60)/tf . measured flow rate ml/min 測量的流速 t . elapsed time to

21、 fill up to mark s 充滿至刻度所花的時間v . volume of the volumetric flask ml 容量瓶的體積  d100×(ff)/fd . deviation % 偏差f . adjusted flow rate ml/min 調(diào)節(jié)后的流速f . measured flow rate ml/min 測量的流速limit限度s: ±5%gradient composition and ripple 梯度組成和波動settings: 設(shè)置stainless steel capillary e.g. 2000 x 0.12 mm

22、installed instead of a column 不銹鋼毛細(xì)管，例如2000x0.12mm，代替色譜柱安裝于儀器detection: uv-detector adjusted to 265 nm 檢測器：紫外檢測器調(diào)節(jié)至265nmmobile phase a: degassed water 流動相a：脫氣水mobile phase b: degassed water containing 0.5% acetone 流動相b：含0.5%的脫氣水flow rate: 1.0 ml/min 流速：1.0ml/分鐘method方法: the test is carried out in th

23、e following way by using a gradient program depending on the number of solvent channels and the configuration of the system: 按下列方法進(jìn)行測試，根據(jù)通道數(shù)量不同使用下列不同系統(tǒng)參數(shù)和梯度進(jìn)行洗脫a-b a-b and a-c a-c, a-b and b-dtime min時間【分鐘】% mobile phase a (water)%流動相a（水相）% mobile phase b%流動相b(water-acetone mixture)（水-丙酮混合物）0.010000

24、.1901010901010.1505020505020.1109030109030.1010040010040.11000start the test by pumping water for at least 10 min to equilibrate the system. 開始測試前，先用水平衡系統(tǒng)至少10分鐘。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 eithe

25、r 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. 開始時的0%值是基線值。所有步驟均在開始時測量基線的水平部分，由軟件或手動在紙上用襯墊打印。100%水/丙酮混合物的高度在以下計算中作為100%值。  %h100* h/h %h . calculated composition 計算組成h . height of t

26、he measured line 測量線的高度h . height of the 100% water/acetone mixture line (mobile phase b) 100%水/丙酮混合物線的高度（流動相b）  d %hg d . deviation 偏差g . gradient composition adjusted % acetone/water solution mixture = mobile phase b 梯度組成調(diào)節(jié)【%丙酮/水溶液混合物 = 流動相b】limit限度s: absolute deviation: ±2 of the adjust

27、ed value 絕對偏差： 調(diào)整值的±2the ripple of the gradient composition is the percentage of noise of the 50% line from the gradient program. 梯度組分的波動為梯度程序噪聲基線的50%  %r 100* n /h50 %r . ripple 波紋h50 . height of the 50% line 50%的線高度n . height of the noise line, measured during 1 minute in the linear regi

28、on 噪聲線高度，在線性區(qū)域1分鐘測量的結(jié)果limit限度s: 0.2 % hplc injector 進(jìn)樣器volume precision and carry-over are the tests proposed for the periodic and motivated check of the hplc injector. 建議在hplc進(jìn)樣器周期性主動檢查中進(jìn)行體積精密度和殘留檢查volume precision and carry-over 體積精密度和殘留solutions: 溶液solvent a: methanol : water r, 60 : 40. 溶液 a：乙醇：

29、水（試劑） = 60：40reference solution (a): dissolve 15.0 mg methyl-, ethyl-, and propylparabene in 100.0 ml of solvent a. 對照液（a）：取15.0mg乙基、甲基和尼泊金丙酯溶于100.0ml溶劑a中reference solution (b): dilute 1.0 ml of reference solution (a) to 10.0 ml of solvent a. 對照液（b）：取1.0ml對照液（a）用溶于10.0ml溶劑a中reference solution (c): d

30、ilute 1.0 ml of reference solution (b) to 100.0 ml of solvent a. 對照液（c）：取1.0ml對照液（b）用溶于100.0ml溶劑a中settings: 設(shè)置column: lichrospher 100 rp8, 5um, 125 x 4 mm, without precolumn 色譜柱：lichrospher 100 rp8, 5 um, 125 x 4 mm, 無襯管mobile phase: methanol : water = 60 : 40 流動相：乙醇：水 = 60：40flow rate: 1.0 ml/min 流

31、動速度：1.0ml/分鐘detection: 254 nm 檢測器：254nminjection volume: 20 l 進(jìn)樣體積：20ulmethod方法: injection scheme: 進(jìn)樣序列 6x reference solution (b) 對照液（b）6針 1x reference solution (a) 對照液（a）1針 1x solvent a (blank injection 1) 溶劑a（空白）1針 1x reference solution (b) 對照液（b）1針 1x solvent a (blank injection 2) 溶劑a（空白2）1針 1x r

32、eference solution (c) 對照液（c）1針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 %.峰面積重復(fù)性：對照液（b）色譜圖中所有峰的峰面積相對標(biāo)準(zhǔn)偏差應(yīng)1.0 %carry-over: the percentage of the peak area correspondi

33、ng 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. 殘留：尼泊金丙酯在空白圖譜中的峰面積不超過在空白后進(jìn)樣的對照液（b）中尼泊金丙酯的對應(yīng)峰面積的10倍的0.5%the percentage of the peak a

34、rea 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. 在對照液（c）中尼泊金丙酯對應(yīng)的峰面積為空白后進(jìn)樣的對照液（b）中尼泊金丙酯峰面積的0.9-1.1%。hplc autosampler 液相色譜儀自動進(jìn)樣器the

35、rmostatting accuracy and precision can be tested in the frame of the periodic and motivated check of the hplc autosampler. hplc自動進(jìn)樣器的周期主動檢查可以進(jìn)行溫度準(zhǔn)確度和精密度檢查thermostatting accuracy 溫度準(zhǔn)確性materials: 材料calibrated temperature probe. 校正過的溫度探頭method方法: select a temperature along the operational or required t

36、emperature range of the equipment. wait until the system is equilibrated. 在可操作范圍或要求的溫度范圍內(nèi)選擇并設(shè)定一個溫度，等待系統(tǒng)平衡by means of the calibrated probe, measure the actual temperature in the autosampler and compare it to the selected temperature. 采用校正過的溫度探頭，測量自動進(jìn)樣器的實(shí)際溫度，與設(shè)定的溫度進(jìn)行比較repeat the same procedure at diff

37、erent pre-selected points covering the temperature range. 在選定的溫度范圍內(nèi)不同溫度點(diǎn)重復(fù)相同的步驟limit限度s: the actual temperature may not differ more than ±3°c with respect to the selected temperature. 實(shí)際溫度與設(shè)定的溫度相比差值不超過±3°cthermostatting precision 溫度準(zhǔn)確性materials: 材料calibrated temperature probe. 校正

38、過的溫度探頭。method方法: select a temperature along the operational or required temperature range of the equipment. wait until the system is equilibrated. 選擇一個操作溫度或要求的儀器溫度范圍，等待系統(tǒng)平衡。by means of the calibrated probe, make “n” measurements over a pre-established period of time. compare the mean of the “n” meas

39、urements to the selected temperature. 采用校正過的溫度探頭，在預(yù)設(shè)的時間內(nèi)進(jìn)行n次測量，比較n次測量值的平均值與設(shè)定的溫度。limits限度: the actual temperature may not differ more than ±3°c with respect to the selected temperature.實(shí)際溫度與設(shè)定的溫度之間差值不超過±3°chplc oven/cooling device 色譜儀柱溫箱/降溫設(shè)施thermostatting accuracy is the paramet

40、er tested in this example of periodic and motivated check of the hplc oven/cooling device. 熱力學(xué)準(zhǔn)確性是在本例對hplc柱溫箱/降溫設(shè)施周期主動檢查的參數(shù)thermostatting accuracy 溫度準(zhǔn)確性materials: 材料calibrated thermometer. 校正過的溫度計method方法: set the column oven temperature to 40 °c, wait about 30 minutes to equilibrate the system

41、, put a calibrated thermometer into the oven and read the temperature after 10 minutes. 設(shè)定柱溫箱溫度為40 °c，等待約30分鐘平衡系統(tǒng)，將校正過的溫度計放入柱溫箱中，在10分鐘后讀取溫度。limits限度: 38 - 42°c. hplc uv/dad detector 紫外/dad檢測器the periodic and motivated check of the hplc uv/dad detector can be performed by testing the linear

42、ity and the wavelength accuracy. 對于hplc紫外/dad檢測器的周期主動檢查，可以對線性和波長準(zhǔn)確性進(jìn)行檢查linearity 線性solutions: 溶劑配制std. 1: 0.5 g caffeine/1 ml methanol hplc grade 標(biāo)準(zhǔn)2： 0.5 g咖啡因/1ml色譜級甲醇std. 2: 1.0 g caffeine/1 ml methanol hplc grade 標(biāo)準(zhǔn)2： 1.0 g咖啡因/1ml色譜級甲醇std. 3: 5.0 g caffeine/1 ml methanol hplc grade 標(biāo)準(zhǔn)3： 5.0 g咖啡因/

43、1ml色譜級甲醇std. 4: 25.0 g caffeine/1 ml methanol hplc grade 標(biāo)準(zhǔn)4：25.0 g咖啡因/1ml色譜級甲醇std. 5: 50.0 g caffeine/1 ml methanol hplc grade 標(biāo)準(zhǔn)5：50.0 g咖啡因/1ml色譜級甲醇std. 6: methanol hplc grade (blank) 標(biāo)準(zhǔn)6：色譜級甲醇（空白）std. 5: weigh 9.0 to 11.0 mg caffeine and fill up to 200.0 ml with methanol hplc 標(biāo)準(zhǔn)5：稱取9.0至11.0mg咖啡因，

44、用hplc級別甲醇稀釋至200.0mlstd. 4: dilute 50.0 ml of std. 5 to 100.0 ml with methanol 標(biāo)準(zhǔn)4：取50.0ml標(biāo)準(zhǔn)5溶液用甲醇稀釋至100.0mlstd. 3: dilute 10.0 ml of std. 5 to 100.0 ml with methanol 標(biāo)準(zhǔn)3：取10.0ml標(biāo)準(zhǔn)溶液5用甲醇稀釋至100.0mlstd. 2: dilute 20.0 ml of std. 3 to 100.0 ml with methanol 標(biāo)準(zhǔn)2：取20.0ml標(biāo)準(zhǔn)3溶液用甲醇稀釋至100.0mlstd. 1: dilute 10

45、.0 ml of std. 3 to 100.0 ml with methanol 標(biāo)準(zhǔn)1：取10.0ml標(biāo)準(zhǔn)3用甲醇稀釋至100.0mlsettings: 設(shè)置column: rp-18 5 um 30-50 x 2,1-4.6 mm or capillary 2000 mm x 0.12 mm id 色譜柱：rp-185 um 30-50 x 2,1-4.6 mm or capillary 2000 mm x 0.12 mm idmobile phase: methanol hplc grade 流動相：色譜級甲醇o(jì)ven temperature: 40 °c 柱溫箱溫度：40

46、°cflow rate: 1.0 ml/min (adjusted by using 100% methanol) 流速：1.0ml/min（用100%甲醇調(diào)整）detection: 273 nm 檢測波長：273nminjection volume: 20l進(jìn)樣量：20ulmethod方法: injection scheme: 進(jìn)樣序列2 x blank 1 x std. 1 1 x std. 2 1 x std. 31 x std. 41 x std. 5 2針空白，標(biāo)準(zhǔn)1-5依序各一針limits限度: r2 0.999 remark: as this test employs

47、 different test solutions to be injected, it covers also the check of correct positioning vials in the autosampler. 注：由于此檢測中要注入不同溶液，因此也包括了自動進(jìn)樣器樣品瓶位置正確性的確認(rèn)wavelength accuracy 波長準(zhǔn)確度if there are built-in test procedures for the determination and adjustment of wavelength accuracy, follow the instruction

48、s of the instrument manual. 如果內(nèi)置檢測程序可以檢測和調(diào)節(jié)波長準(zhǔn)確度，則根據(jù)操作手冊進(jìn)行操作in all other cases use the procedure described below. 在所有其它情況下采用以下程序solutions: 溶液dad: caffeine std. 5 from the linearity testing 線性測試中咖啡因標(biāo)準(zhǔn)溶液5號uv/vis: 1.0 g/ml anthracene solution in water 蒽水溶液settings: 設(shè)置mobile phase: 15% acetonitrile in w

49、ater流動相：15%乙腈水溶液column: rp18, 5 mm 30-50 x 2,1-4.6 mm or capillary 2.0 m x 0.12 mm id 色譜柱：rp18，5m, 30-50×2.1-4.6mm或毛細(xì)管柱20m×0.12mm內(nèi)徑oven temperature: 40 °c 柱箱溫度：40flow rate: 1.0 ml/min (adjusted by using 15% acetonitrile in water) 流速：1.0ml/min（用15%的乙腈水溶液調(diào)節(jié)）detection: scan from 230 nm t

50、o 290 nm (dad) 檢測：自230至290nm掃描（dad）injection volume: 20 l 進(jìn)樣體積：20lmethod方法: dad: inject 20l of the caffeine solution and record the spectrum. the maximum is at 272 nm and the minimum at 244 nm. dad: 咖啡因溶液進(jìn)樣20l，記錄色譜圖，最大吸收波長為272nm，最小為244nm.uv/vis: fill the cell with the anthracene solution and change

51、the wavelength from 248 to 254 nm in 1 nm steps; record the maximum of absorption. the theoretical value is 251 nm. uv/可見光：向池中注入蒽溶液，將波長以1nm的步距從248變化至254nm，記錄最大吸收值。理論值為251nm。limit限度s: 2 nm hplc fluorescence detector 熒光檢測器the following three parameters are proposed for the performance of the periodic

52、and motivated check of the hplc fluorescent detector:下面三個參數(shù)建議作為熒光檢測器性能周期自主檢查wavelength accuracy excitation 波長激發(fā)準(zhǔn)確度method方法: 方法rinse and fill the measuring cell with de-ionized water 用去離子水淋洗并充滿檢測池adjust the excitation wavelength to 350 nm. 調(diào)節(jié)激發(fā)波長為350nmmeasure the emission and subtract 397 nm (theoret

53、ical value). 測量發(fā)射波長減去397nm（理論值）limit限度限度s: ±3 nm wavelength accuracy emission 波長發(fā)射準(zhǔn)確度method方法: 方法rinse and fill the measuring cell with de-ionized water用去離子水淋洗并充滿檢測池adjust the emission wavelength to 397 nm. 調(diào)節(jié)發(fā)射波長為397nmmeasure the excitation and subtract 350 nm (theoretical value). 測量激發(fā)波長減去350n

54、m（理論值）limit限度限度s:±3nm sensitivity 靈敏度solutions: 溶液quinine hcl.2h2o solution conc. 0.015g/ml (=15 ppb) 二水鹽酸喹啉溶液，濃度0.015g/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

55、, 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. 喹啉溶液用以下流動相制備：溶解6.8g磷酸二氫鉀和3.0g己胺于700ml試劑級水中，用稀磷酸調(diào)ph至2.8，加90ml乙腈并用試劑級水稀釋至1000.0ml.settings: 設(shè)置the chromatographic conditions are set according to ph. eur. “quinine hcl” (01/2005:0018), t

56、est “other cinchona alkaloids”, with modified flow rate and acetonitrile concentration. 色譜條件設(shè)定依據(jù)歐洲藥典“鹽酸喹啉”（01/2005:0018），檢測“金雞納生物堿”，改變流速和乙腈濃度。mobile phase: as above 流動相：同上column: rp18, 5 m, 250 × 4.6 mm 色譜柱: rp18, 5 m, 250 ×4.6 mm flow rate: 1.2 ml/min 流速：1.2 ml/minexcitation wavelength: 350 nm 激發(fā)波長：350nmemission wavelength: 397 nm 發(fā)射波長：397nmflow-cell volume: 8 l (for this example,