HPLC法在國內(nèi)外藥典中的應(yīng)用與比較課件

1、HPLC法在國內(nèi)外藥典中的應(yīng)用與比較2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較HPLC法在國內(nèi)外藥典中的應(yīng)用與比較2022/10/9HPL匯報(bào)的主要內(nèi)容 一、簡述 二、高效液相色譜儀 三、系統(tǒng)適用性 四、色譜條件的調(diào)整 五、HPLC的應(yīng)用及方法開發(fā)2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較2匯報(bào)的主要內(nèi)容 一、簡述2022/10/9HPLC法在國內(nèi)外一、簡述中國藥典2010年版二部共收載2271個(gè)品種，新增品種330個(gè)，修訂品種1500個(gè)，涉及HPLC檢測項(xiàng)目的品種有1291個(gè)，占總品種的57%，其中新增/修訂926個(gè)。HPLC法在整個(gè)藥典品種的檢驗(yàn)中占有重要地位。

2、2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較3一、簡述中國藥典2010年版二部共收載2271個(gè)品種，新High Performance Liquid Chromatography簡稱HPLC，開始應(yīng)用于20世紀(jì)60年代后期，現(xiàn)已趨于成熟，廣泛應(yīng)用于醫(yī)藥、生化、天然產(chǎn)物主要組分分析，以及食品、化妝品分析，環(huán)境分析，農(nóng)業(yè)分析，石油化工分析等。優(yōu)點(diǎn)：高壓+高速+高效+高靈敏度一、簡述2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較4High Performance Liquid ChromaCHP(2010)：中國藥典2010年版USP：United Stated Pharmac

3、opoeia EP：European PharmacopoeiaJP：Japanese Pharmacopoeia一、簡述2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較5CHP(2010)：中國藥典2010年版一、簡述2022定義CHP(2010)附錄V D：HPLC系采用高壓輸液泵將規(guī)定的流動相泵入裝有填充劑的色譜柱，對供試品進(jìn)行分離測定的色譜方法。USP(32)：High-pressure liquid chromatography (HPLC), sometimes called high-performance liquid chromatography, is a sepa

4、ration technique based on a solid stationary phase and a liquid mobile phase. Separations are achieved by partition, adsorption, or ion-exchange processes, depending upon the type of stationary phase used.2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較6定義CHP(2010)附錄V D：HPLC系采用高壓輸液泵將定義EP(6.0) 2.2.29：Liquid chromatograp

5、hy is a method of chromatographic separation based on the difference in the distribution of species between two non-miscible phases, in which percolates through a stationary phase contained in a column.2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較7定義EP(6.0) 2.2.29：Liquid chroma定義JP(XV)2.01：Liquid chromatography is a

6、 method to develop a mixture injected into a column prepared with a suitable stationary phase by passing a liquid as a mobile phase through the column, in order to separate the mixture into its components by making use of the difference of retention capacity against the stationary phase, and to dete

7、rmine the components.2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較8定義JP(XV)2.01：Liquid chromatogr二、高效液相色譜儀基本組成：2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較9二、高效液相色譜儀基本組成：2022/10/9HPLC法在國泵CHP(2010)USP(32)EP(6.0)JP(XV) 未解釋滿足等度和梯度洗脫;壓力：5000psi或更高;最大可達(dá)10ml/min可滿足等度或梯度洗脫只強(qiáng)調(diào)了恒定速率泵的種類很多，目前應(yīng)用最多的是柱塞往復(fù)泵（恒流泵）2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較10泵CHP

8、(2010)USP(32)EP(6.0)JP(XV)進(jìn)樣器CHP(2010)USP(32)EP(6.0)JP(XV)未解釋微量進(jìn)樣器定量環(huán)自動進(jìn)樣器手動 / 自動強(qiáng)調(diào)重復(fù)性好即可2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較11進(jìn)樣器CHP(2010)USP(32)EP(6.0)JP(X色譜柱分離的核心CHP(2010)附錄：1.正相：硅膠柱； 反相： C-18柱、C-8柱；2.粒徑：普通310m； 2m（亞-2m僅能用于UPLC）；3.溫度要求：以硅膠為載體的通常 40；不宜超過60 ；4.pH要求：28；8，硅膠溶解以5m最為常見2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用

9、與比較12色譜柱分離的核心CHP(2010)附錄：2，化學(xué)鍵合相脫色譜柱技術(shù)發(fā)展很快，新填料新技術(shù)也不斷涌現(xiàn)，給我們提供了更多的選擇。Gemini-NX C18柱：色譜柱乙烷交聯(lián)，抵抗高pH侵蝕多點(diǎn)配體鍵合，抵抗低pH下配體水解pH112穩(wěn)定！Kinetex C18柱：通過核-殼顆粒技術(shù)，使顆粒減小到2.6m，而無需高壓即可達(dá)到提高速度、分離度和靈敏度的效果。2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較13色譜柱技術(shù)發(fā)展很快，新填料新技術(shù)也不斷涌現(xiàn)，給我們提供了更多色譜柱Zorbax StableBond柱：采用了較大的二異丁基(SB-C18)或二異丙基(SB-C8、SB-C3、S

10、B-Phenyl、SB-CN、SB-Aq)側(cè)鏈基團(tuán)和空間位阻，避免了在低pH條件下的水解破壞，其pH1.08.0，溫度上限也可達(dá)到8090，甚至在100%水相中也有出色的表現(xiàn)。2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較14色譜柱Zorbax StableBond柱：2022/10/色譜柱Zorbax Extend C18柱：采用獨(dú)特的雙配位C18-C18鍵合技術(shù)，使的在高pH條件下使用硅膠基色譜柱成為可能，在pH211.5的范圍內(nèi)是穩(wěn)定的。2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較15色譜柱Zorbax Extend C18柱：2022/10/USP(32):Sta

11、tionary phases for modern, reverse-phase liquid chromatography typically consist of an organic phase chemically bound to silica or other materials.1. Particles：usually 3 to 10 m in diameter；may range up to 50 m or more for preparative columns. 2. Internal diameters：usually 2 to 5mm for analytical se

12、paration，lager for preparative chromatography.3. Temperature: only rarely above 60 ；4.根據(jù)填料不同，USP在Chromatographic reagents in the Reagents, Indicators, and Solutions section列出了USP-NF所用到的不同類型的柱子，如下所示:2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較16USP(32):Stationary phasesC-18硅膠柱C-8NH2柱陽離子交換柱（SCX）CN柱苯基柱陰離子交換柱（SAX）2022/

13、10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較17C-18硅膠柱C-8NH2柱陽離子交換柱CN柱苯基柱陰離子交L57L67未列出2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較18L57L67未列出2022/10/9HPLC法在國內(nèi)外藥典EP(6.0) 2.2.29： 大部分分離機(jī)制都是基于以化學(xué)鍵合硅膠作為固定相，極性溶劑作為流動相的色譜條件。而化學(xué)鍵合相的性質(zhì)往往決定了色譜系統(tǒng)的分離性能。2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較19EP(6.0) 2.2.29： 大部分分離機(jī)制都是基于以化學(xué)particle size：310m internal diameters：

14、prescribed in the monograph, eg:pH：silica based reversed-phase columns are considered to be stable in mobile phases having an apparent pH in the range 2.0 to 8.0.temperature: not be heated above 60 Special：EP(6.0) 2.2.29：stationary phase degradetioncomposition of the mobile phase2022/10/10HPLC法在國內(nèi)外藥

15、典中的應(yīng)用與比較20particle size：310m EP(6.0) 2EPMannitol Assay:2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較21EPMannitol Assay:2022/10/9HPLCJP(XV)：A column with a stationary phase chemically bound on the inside wall instead of the column packed with the packing material may be used.2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較22JP(XV)：A col

16、umn with a station檢測器CHP(2010):UVDADFLDECDELSDRIDMS優(yōu)點(diǎn)：靈敏度高、噪音低、線性范圍寬、對流速和溫度的波動不靈敏，適用于梯度洗脫及制備色譜。 缺點(diǎn)：只能檢測有紫外吸收的物質(zhì)，流動相的選擇有一定限制，流動相的截止波長必須小于檢測波長。 適用范圍：大多數(shù)有紫外吸收的化合物。優(yōu)點(diǎn)：通用型檢測器，對各種物質(zhì)有幾乎相同的響應(yīng)。缺點(diǎn)：靈敏度相對較低，流動相必須是揮發(fā)性的，不能用非揮發(fā)性的緩沖鹽及表面活性劑。適用范圍：適用于揮發(fā)性低于流動相的組分，主要用于糖類、高級脂肪酸、磷脂、維生素、氨基酸、甘油三酯、皂苷及甾體等等無紫外吸收或紫外末端吸收的化合物的檢測。

17、 優(yōu)點(diǎn)：靈敏度高、選擇性好，是微量組分和體內(nèi)藥物分析常用的檢測器之一。 缺點(diǎn)：只適用于能夠產(chǎn)生熒光的物質(zhì)的檢測，適用范圍不如紫外檢測器。影響因素較多。 適用范圍：具有天然熒光的物質(zhì)或通過熒光衍生化變成熒光衍生物后測定，主要用于氨基酸、多環(huán)芳烴、維生素、甾體化合物及酶等的檢測。優(yōu)點(diǎn)：靈敏度很高，尤其適用于痕量組分分析。缺點(diǎn)：干擾比較多，對溫度和流速的變化比較敏感。適用范圍：應(yīng)用范圍廣，凡具氧化還原活性的或經(jīng)衍生化后具氧化還原活性的物質(zhì)。優(yōu)點(diǎn)：靈敏度高，選擇性好，能同時(shí)給出組分的結(jié)構(gòu)信息。缺點(diǎn)：響應(yīng)信號受離子化效率限制，儀器較為昂貴，流動相必須是揮發(fā)性的，不能用非揮發(fā)性的緩沖鹽及表面活性劑。適用范

18、圍：組分的結(jié)構(gòu)鑒別，微量及痕量組分的分析，藥物代謝分析等。優(yōu)點(diǎn)：通用型檢測器，只要組分的折光率與流動相的折光率有足夠的差別就能檢測。缺點(diǎn)：靈敏度低、受環(huán)境溫度、流量及流動相組成等波動的影響大，一般不能用于梯度洗脫。適用范圍：RID為通用型檢測器，適用于無紫外吸收化合物的分析，如糖類分析。2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較23檢測器CHP(2010):優(yōu)點(diǎn)：靈敏度高、噪音低、線性范圍寬USP and EPUSP(32)EP(6.0)Fixed, variable and multi-wavelength detectors are widely available.UV、R

19、ID、FLD、ECD.New detectors continue to be developed in attempts to overcome the deficiencies of those being used.UV/Vis spectrophotometers, including diode array detectors, are the most commonly employed detectors.2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較24USP and EPUSP(32)EP(6.0)Fixed,流動相CHP(2010)反相系統(tǒng)首選甲醇-水系統(tǒng)（采用紫

20、外末端波長檢測，首選乙腈-水系統(tǒng)）緩沖鹽：少用，盡可能低反相色譜系統(tǒng)，C-18柱，有機(jī)相比例應(yīng)不低于5%。USP(32)High-purity reagents and “HPLC grade”organic solventswater: low condutivity and low UV absorptionEP(6.0)For nomal-phase chromatography, less polar solvents are employed. Water is to be strictly controlled!0.45mdegassed by sparing with heliu

21、m,sonication or using on-line membrane/vacuumAdjustment of the pH,is effected using only the aqueous component of the mobile phase and not the mixture.2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較25流動相CHP(2010)反相系統(tǒng)首選甲醇-水系統(tǒng)（采用紫外末系統(tǒng)適用性System Suitability2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較26系統(tǒng)適用性2022/10/9HPLC法在國內(nèi)外藥典中的應(yīng)用與定義CHP(

22、2010):色譜系統(tǒng)的適用性試驗(yàn)通常包括理論板數(shù)、分離度、重復(fù)性和拖尾因子四個(gè)參數(shù)，其中，分離度和重復(fù)性尤為重要。ICH definition: System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system th

23、at can be evaluated as such. 2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較27定義CHP(2010):色譜系統(tǒng)的適用性試驗(yàn)通常包括理論板數(shù)DefinitionEP (6.0): The system suitability tests represent an integral part of the method and are used to ensure adequate performance of the chromatographic system. The various components of the equipment employ

24、ed must be qualified and be capable of achieving the precision required to conduct the test or assay. USP(32): System suitability tests are an integral part of gas and liquid chromatographic methods. They are used to verify that the resolution and reproducibility of the chromatographic system are ad

25、equate for the analysis to be done.2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較28DefinitionEP (6.0): The systemDefinitionUSP(32): No sample analysis is acceptable unless the requirements of system suitablity have been met. Sample analyses obtained while the system fails requirements are unacceptable. System suitabi

26、lity must be demonstrated throughout the run by injection of an appropriate control preparation at appropriate intervals. Wherever there is a significant change in equipment or in a critical reagent, suitability testing should be performed before the injection of samples.2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較

27、29DefinitionUSP(32): No sample 理論板數(shù)評價(jià)色譜柱的重要指標(biāo)。CHP(2010) *USP(32) *EP(6.0)JP(XV)n=16(tR/W)2 n=5.54(tR/Wh/2)2N=16(t/W)2N=5.54(t/Wh/2)2N=5.54(tR/Wh)2Wh：半高峰寬N=5.54(tR/W0.5h)2W0.5h：半高峰寬 影響因素：固定相、柱溫、流動相和保留時(shí)間。*有爭議時(shí)，以峰寬（W）計(jì)算結(jié)果為準(zhǔn)2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較30理論板數(shù)評價(jià)色譜柱的重要指標(biāo)。CHP(2010) *USP(分離度（R）衡量色譜系統(tǒng)效能的關(guān)鍵指標(biāo)！

28、 2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較31分離度（R）衡量色譜系統(tǒng)效能的關(guān)鍵指標(biāo)！ 2022/10/9分離度（R）CHP(2010)*R=2(tR2-tR1)/(W1+W2)R=2(tR2-tR1)/1.70(W1,h/2+W2,h/2)除另有規(guī)定外，應(yīng)大于1.5 USP(32) *# EP(6.0) # JP(XV)* 有爭議時(shí)，以峰寬（W）計(jì)算結(jié)果為準(zhǔn)；# described in individual monograph2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較32分離度（R）CHP(2010)*R=2(tR2-tR1)/(USP(32)Chromato

29、graphic system The liquid chromatograph is equipped with a 275-nm detector and a 4.6-mm 15-cm column that contains packing L1 the resolution, R, between the impurity C and famotidine peaks is not less than 1.3; the resolution, R, between the famotidine and impurity D peaks is not less than 1.3;Famot

30、idine TabletsAssary 2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較33USP(32)Chromatographic system EP(6.0)左甲狀腺素鈉含量測定：左羥丙哌嗪對映體純度：2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較34EP(6.0)左甲狀腺素鈉含量測定：左羥丙哌嗪對映體純度：2重復(fù)性 評價(jià)連續(xù)進(jìn)樣中，色譜系統(tǒng)響應(yīng)值的重復(fù)性能。 CHP(2010):外標(biāo)法：對照品溶液，連續(xù)進(jìn)樣5次，峰面積RSD不得過2.0%。內(nèi)標(biāo)法：配制相當(dāng)于80%、100%、120%的對照品溶液，加入內(nèi)標(biāo)溶液，分別至少進(jìn)樣2次，計(jì)算平均校正因子的RSD不得過2.0%

31、 。2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較35重復(fù)性 評價(jià)連續(xù)進(jìn)樣中，色譜系統(tǒng)響應(yīng)值的重復(fù)性能。202重復(fù)性 USP(32):Unless otherwise specified in the individual monograph , data from five replicate injections of the analyte are used to calculate the relative standard deviation, SR, if the requirment is 2.0% or less; data from six replicate in

32、jections are used if the relative standard deviation requirment is more than 2.0%. 2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較36重復(fù)性 USP(32):2022/10/9HPLC法在國內(nèi)重復(fù)性 EP(6.0):The repeatability of response is expressed as an es timated percentage relative standard deviation(Sr(%) of a consecutive series of measurements f

33、or NOT fewer than 3 injections or applications of a reference solution.mean of individual valuesindividual values expressed as peak area, peak height, or ratio of areas by the interal standardisation methodnumber of individual values2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較37重復(fù)性 EP(6.0):mean of individu重復(fù)性 EP(6.

34、0):Unless otherwise prescribed, the maximum permitted RSD does not exceed the appropriate value given in table.Number of individual injections3456B(percent)Maximum permitted relative standard deviation2.00.410.590.730.852.50.520.740.921.063.00.620.891.101.272022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較38重復(fù)性 EP(6.0):

35、Number of indivi重復(fù)性NOTE: This requirement does not apply to tests for related substances.upper limit given in the definition of the individual monograph minus 100%contant(0.349)number of replicate injections of the reference solution (3n6)90% probability level，n-1 degrees of freedomEP(6.0): In an as

36、say of an active substance where the value is 100 percent for a pure substance, the maximum permitted (Sr(%)max) for defined limits is calculated using the following equation:2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較39重復(fù)性NOTE: This requirement does重復(fù)性 JP(XV): 公式與EP相同。重復(fù)次數(shù)及限度在各論中要求。例： Ritodrine Hydrochloride Rela

37、ted subsances System suitability Test for required detectability: . System performance:. System repeatability: When the test is repeated 6 times with .，the relative standard deviation of the peak areas of deferoxamine is not more than 3.0%.2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較40重復(fù)性 JP(XV): 公式與EP相同。2022/10/拖尾

38、因子（對稱因子）用于評價(jià)色譜峰的對稱性2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較41拖尾因子（對稱因子）用于評價(jià)色譜峰的對稱性2022/10/9拖尾因子T（or Symmetry factor）CHP(2010)T=W0.05h/2d1應(yīng)符合個(gè)論項(xiàng)下的規(guī)定除另有規(guī)定外，峰高法定量時(shí)T應(yīng)在0.951.05之間USP(32)T=W0.05/2fEP(6.0)As=W0.05h/2dAn As value of 1.0 signifies symmetry. When As 1.0, the peak is tailing. When As 1.0, the peak is front

39、ing.In a related substances test or assay, for a peak in the chromatogram obtained with a reference solution used for quantification, the symmetry factor is 0.8 to 1.5, unless otherwise prescribed.JP(XV)S=W0.05h/2f2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較42拖尾因子T（or Symmetry factor）CHP(2USPIt is also a common pra

40、ctice to measure the Asymmetry factor as the ratio of the distance between the vertical line connecting the peak apex with the interpolated baseline and the peak front, and the distance between that line and the peak back measure at 10% of the peak height, it would be (W0.10-f0.10)/f0.10However, for

41、 the purpose of USP, only the formula presented in the Glossary of Symbols is valid. 2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較43USPIt is also a common practicEP中系統(tǒng)適用性：p/v and S/NThe peak-to-valley ratio (p/v) may be employed as a system suitability criterion in a test for related substances when baseline separat

42、ion between 2 peaks is not achieved.p/v=Hp/HvFor example: Econazole related substances2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較44EP中系統(tǒng)適用性：p/v and S/NThe peak-tEPThe short-term noise influences the precision of quantification.The signal-to-noise ratio is calculated using the following equation:S/N=2H/h For exampl

43、e: ketotifen hydrogen fumarate related substances2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較45EPThe short-term noise influen色譜條件的調(diào)整Adjustment of Chromatographic Conditions2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較46色譜條件的調(diào)整2022/10/9HPLC法在國內(nèi)外藥典中的應(yīng)CHP(2010)明確規(guī)定：不可變的有：固定相的種類、流動相的組分、檢測器類型可變的有：色譜柱內(nèi)徑、長度、載體粒度、流動相流速、混合流動相各組分的比例、柱溫、進(jìn)樣量、檢

44、測器的靈敏度。2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較47CHP(2010)明確規(guī)定：2022/10/9HPLC法在國CHP(2010)與CHP(2005)不同：中國藥典2010年版規(guī)定了流動相調(diào)整的限度。調(diào)整流動相組分比例時(shí)，以組分比例較低者（50）相對于自身改變量不超過30且相對于總量的改變量不超過10為限，如30相對改變量的數(shù)值超過總量的10時(shí)，則改變量以總量的10為限。 2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較48CHP(2010)與CHP(2005)不同：中國藥典20CHP(2010)例：奧美拉唑腸溶片，釋放度檢查，色譜條件：問題：峰型差！流動相可調(diào)范

45、圍：82.5:17.5 67.5:32.5解決：70：302022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較49CHP(2010)例：奧美拉唑腸溶片，釋放度檢查，色譜條件：USP(32)pH of mobile phaseconcentration of saltsin bufferratio of components in mobile phaseWavelength of UV detectorinjection volumepH of the aqueous buffer within 0.2 units of the value or range specified withi

46、n 10%provided the permitted pH variation is metapply to minor components can be adjusted by 30 relativecan not exceed 10% absolute, see example:binary or ternary mixturesnot permittedcan be reduced as far as is consistent with accepted precision and detection limitscolumn lengthcolumn inner diameter

47、particle sizeflow ratecolumn temperature70%25%can be reduced by as much as 50%50%10 If adjustments of operating conditions to meet system suitability requirments are necessary, each of the following is the maxium variation that can be considered, unless otherwise directed in the monograph.2022/10/10

48、HPLC法在國內(nèi)外藥典中的應(yīng)用與比較50USP(32)pH of mobile phaseconceUSP(32)Binary Mixtures SPECIFIED RATIO OF 50:50 Thirty percent of 50 is 15% absolute, but this exceeds the maximum permitted change of 10% absolute in either component. Therefore, the mobile phase ratio may be adjusted only within the range of 40:60

49、to 60:40. SPECIFIED RATIO OF 2:98 Thirty percent of 2 is 0.6% absolute. Therefore the maximum allowed adjustment is within the range of 1.4:98.6 to 2.6:97.4. 2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較51USP(32)Binary Mixtures 2022/USP(32)Ternary Mixtures SPECIFIED RATIO OF 60:35:5 For the second component, 30% of

50、35 is 10.5% absolute, which exceeds the maximum permitted change of 10% absolute in any component. Therefore the second component may be adjusted only within the range of 25% to 45% absolute. For the third component, 30% of 5 is 1.5% absolute. In all cases, a sufficient quantity of the first compone

51、nt is used to give a total of 100%.Therefore, mixture ranges of 50:45:5 to 70:25:5 or 58.5:35:6.5 to 61.5:35:3.5 would meet the requirement. 2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較52USP(32)Ternary Mixtures 2022EP(6.0)The extent to which the various parameters of a chromatographic test may be adjusted to satisf

52、y the system suitability criteria without fundamentally modifying the methods are listed below.Isocratic elution :pH of the aqueous component of the mobile phaseconcentration of salts in buffercomposition of the mobile phaseWavelengthcolumn temperature0.2pH 1.0pH (non-ionisable) within 10%Minor comp

53、onent 30% relative or 2% absolute whichever is larger.No component can be altered by more than 10% absolute （see example）no adjustment permitted10column lengthinteral diameterparticle sizeflow rateinjection volume70%25%maximum reduction of 50%no increase permitted50% a larger adjustment is acceptabl

54、e（see example）may be decreasedno increase permitted2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較53EP(6.0)The extent to which theEP(6.0)Composition of the mobile phase: the amount of the minor solvent component may be adjusted by 30 % relative or 2 % absolute, whichever is the larger; for a minor component at 10 % of

55、 the mobile phase, a 30 % relative adjustment allows a range of 7-13% wherea 2% absolute adjustment allows a range of 8-12%, the relative value being therefore the larger;For a minor component at 5% of the mobile phase, a 30% relative adjustment allows a range of 3.5-6.5% whereas a 2% absolute adjus

56、tment allows a range of 3-7%, the absolute value being in this case the larger.No other component is altered by more than 10% absolute.2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較54EP(6.0)Composition of the mobiEP(6.0)When column dimensions are changed,the flow rate may be ajusted as necessary using equation:F1：規(guī)定的

57、流速F2：調(diào)整后的流速l1：規(guī)定的柱長l2：實(shí)際所用的柱長d1：規(guī)定柱子的內(nèi)徑d2：實(shí)際所用柱子的內(nèi)徑2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較55EP(6.0)When column dimensions EP(6.0)pH of the aqueous component of the mobile phaseconcentration of salts in buffercomposition of the mobile phaseWavelength of UV detectorcolumn temperatureno adjustmentpermitted no adj

58、ustment permittedthe system suitability requirements are fulfilleldthe principal peak elute within 15% of the indicated retention timeelution power is not weaker than beforeno adjustment permitted5column lengthinteral diameterparticle sizeflow rateinjection volume70%25%no adjustment permittedsame as

59、isocratic elution may be decreasedno increase permittedGradient elution：more critical than with isocratic elution2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較56EP(6.0)pH of the aqueous conceHPLC的應(yīng)用及方法開發(fā)2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較57HPLC的應(yīng)用及方法開發(fā)2022/10/9HPLC法在國內(nèi)外HPLC應(yīng)用類型定 義I原料藥或藥物制劑活性成分中主要成分的分析程序-如含量測定II原料藥中雜質(zhì)檢查或藥物制劑

60、中降解產(chǎn)物的分析程序，這些分析程序包括定量試驗(yàn)或限度試驗(yàn)III用于性能參數(shù)測試的分析程序-如溶出度、釋放度IV鑒別試驗(yàn)HPLC 可以用來干什么？2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較58HPLC應(yīng)用類型定 義I原料藥或藥物制劑活性成分中主要成分方法開發(fā)的指導(dǎo)原則ICHUSP 中國藥典2022/10/10HPLC法在國內(nèi)外藥典中的應(yīng)用與比較59方法開發(fā)的指導(dǎo)原則ICH2022/10/9HPLC法在國內(nèi)外ICHICH(人用藥品注冊技術(shù)要求國際協(xié)調(diào)會)三方協(xié)調(diào)指導(dǎo)原則 Q2A：分析方法論證的文本ICH三方協(xié)調(diào)指導(dǎo)原則 Q2B：方法學(xué)ICH三方協(xié)調(diào)指導(dǎo)原則2022/10/10HPLC