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1、HPLC法在国内外药典中的应用与比较,2022/11/4,HPLC法在国内外药典中的应用与比较,HPLC法在国内外药典中的应用与比较2022/10/9HPL,汇报的主要内容,一、简述 二、高效液相色谱仪 三、系统适用性 四、色谱条件的调整 五、HPLC的应用及方法开发,2022/11/4,HPLC法在国内外药典中的应用与比较,2,汇报的主要内容 一、简述2022/10/9HPLC法在国内外,一、简述,中国药典2010年版二部共收载2271个品种，新增品种330个，修订品种1500个，涉及HPLC检测项目的品种有1291个，占总品种的57%，其中新增/修订926个。HPLC法在整个药典品种的检验
2、中占有重要地位。,2022/11/4,HPLC法在国内外药典中的应用与比较,3,一、简述中国药典2010年版二部共收载2271个品种，新,High Performance Liquid Chromatography简称HPLC，开始应用于20世纪60年代后期，现已趋于成熟，广泛应用于医药、生化、天然产物主要组分分析，以及食品、化妆品分析，环境分析，农业分析，石油化工分析等。优点：高压+高速+高效+高灵敏度,一、简述,2022/11/4,HPLC法在国内外药典中的应用与比较,4,High Performance Liquid Chroma,CHP(2010)：中国药典2010年版USP：Unit
3、ed Stated Pharmacopoeia EP：European PharmacopoeiaJP：Japanese Pharmacopoeia,一、简述,2022/11/4,HPLC法在国内外药典中的应用与比较,5,CHP(2010)：中国药典2010年版一、简述2022,定义,CHP(2010)附录V D：HPLC系采用高压输液泵将规定的流动相泵入装有填充剂的色谱柱，对供试品进行分离测定的色谱方法。USP(32)：High-pressure liquid chromatography (HPLC), sometimes called high-performance liquid ch
4、romatography, is a separation 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.,2022/11/4,HPLC法在国内外药典中的应用与比较,6,定义CHP(2010)附录V D：HPLC系采用高压输液泵将,定义,EP(6.0
5、) 2.2.29：Liquid chromatography 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.,2022/11/4,HPLC法在国内外药典中的应用与比较,7,定义EP(6.0) 2.2.29：Liquid chroma,定义,JP(X
6、V)2.01：Liquid chromatography is a 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
7、the stationary phase, and to determine the components.,2022/11/4,HPLC法在国内外药典中的应用与比较,8,定义JP(XV)2.01：Liquid chromatogr,二、高效液相色谱仪,基本组成：,2022/11/4,HPLC法在国内外药典中的应用与比较,9,二、高效液相色谱仪基本组成：2022/10/9HPLC法在国,泵,泵的种类很多，目前应用最多的是柱塞往复泵（恒流泵）,2022/11/4,HPLC法在国内外药典中的应用与比较,10,泵CHP(2010)USP(32)EP(6.0)JP(XV),进样器,2022/11/4,
8、HPLC法在国内外药典中的应用与比较,11,进样器CHP(2010)USP(32)EP(6.0)JP(X,色谱柱分离的核心,CHP(2010)附录：1.正相：硅胶柱； 反相： C-18柱、C-8柱；2.粒径：普通310m； 2m（亚-2m仅能用于UPLC）；3.温度要求：以硅胶为载体的通常 40；不宜超过60 ；4.pH要求：28；,8，硅胶溶解,以5m最为常见,2022/11/4,HPLC法在国内外药典中的应用与比较,12,色谱柱分离的核心CHP(2010)附录：2，化学键合相脱,色谱柱技术发展很快，新填料新技术也不断涌现，给我们提供了更多的选择。Gemini-NX C18柱：,色谱柱,Ki
9、netex C18柱：通过核-壳颗粒技术，使颗粒减小到2.6m，而无需高压即可达到提高速度、分离度和灵敏度的效果。,2022/11/4,HPLC法在国内外药典中的应用与比较,13,色谱柱技术发展很快，新填料新技术也不断涌现，给我们提供了更多,色谱柱,Zorbax StableBond柱：采用了较大的二异丁基(SB-C18)或二异丙基(SB-C8、SB-C3、SB-Phenyl、SB-CN、SB-Aq)侧链基团和空间位阻，避免了在低pH条件下的水解破坏，其pH1.08.0，温度上限也可达到8090，甚至在100%水相中也有出色的表现。,2022/11/4,HPLC法在国内外药典中的应用与比较,1
10、4,色谱柱Zorbax StableBond柱：2022/10/,色谱柱,Zorbax Extend C18柱：采用独特的双配位C18-C18键合技术，使的在高pH条件下使用硅胶基色谱柱成为可能，在pH211.5的范围内是稳定的。,2022/11/4,HPLC法在国内外药典中的应用与比较,15,色谱柱Zorbax Extend C18柱：2022/10/,USP(32):,Stationary phases for modern, reverse-phase liquid chromatography typically consist of an organic phase chemical
11、ly 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 separation，lager for preparative chromatography.3. Temperature: only rarely above 60 ；4.根据填料不同，USP在Chromatograp
12、hic reagents in the Reagents, Indicators, and Solutions section列出了USP-NF所用到的不同类型的柱子，如下所示:,2022/11/4,HPLC法在国内外药典中的应用与比较,16,USP(32):Stationary phases,C-18,硅胶柱,C-8,NH2柱,阳离子交换柱（SCX）,CN柱,苯基柱,阴离子交换柱（SAX）,2022/11/4,HPLC法在国内外药典中的应用与比较,17,C-18硅胶柱C-8NH2柱阳离子交换柱CN柱苯基柱阴离子交,L57L67未列出,2022/11/4,HPLC法在国内外药典中的应用与比较,
13、18,L57L67未列出2022/10/9HPLC法在国内外药典,EP(6.0) 2.2.29：,大部分分离机制都是基于以化学键合硅胶作为固定相，极性溶剂作为流动相的色谱条件。而化学键合相的性质往往决定了色谱系统的分离性能。,2022/11/4,HPLC法在国内外药典中的应用与比较,19,EP(6.0) 2.2.29： 大部分分离机制都是基于以化学,particle size：310m internal diameters：prescribed in the monograph, eg:pH：silica based reversed-phase columns are considered
14、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：,2022/11/4,HPLC法在国内外药典中的应用与比较,20,particle size：310m EP(6.0) 2,EP,Mannitol Assay:,2022/11/4,HPLC法在国内外药典中的应用与比较,21,EPMannitol Assay:2022/10/9HPLC,JP(XV)：,A column wit
15、h a stationary phase chemically bound on the inside wall instead of the column packed with the packing material may be used.,2022/11/4,HPLC法在国内外药典中的应用与比较,22,JP(XV)：A column with a station,检测器,CHP(2010):UVDADFLDECDELSDRIDMS,优点：灵敏度高、噪音低、线性范围宽、对流速和温度的波动不灵敏，适用于梯度洗脱及制备色谱。 缺点：只能检测有紫外吸收的物质，流动相的选择有一定限制，流动相的
16、截止波长必须小于检测波长。 适用范围：大多数有紫外吸收的化合物。,优点：通用型检测器，对各种物质有几乎相同的响应。缺点：灵敏度相对较低，流动相必须是挥发性的，不能用非挥发性的缓冲盐及表面活性剂。适用范围：适用于挥发性低于流动相的组分，主要用于糖类、高级脂肪酸、磷脂、维生素、氨基酸、甘油三酯、皂苷及甾体等等无紫外吸收或紫外末端吸收的化合物的检测。,优点：灵敏度高、选择性好，是微量组分和体内药物分析常用的检测器之一。 缺点：只适用于能够产生荧光的物质的检测，适用范围不如紫外检测器。影响因素较多。 适用范围：具有天然荧光的物质或通过荧光衍生化变成荧光衍生物后测定，主要用于氨基酸、多环芳烃、维生素、甾
17、体化合物及酶等的检测。,优点：灵敏度很高，尤其适用于痕量组分分析。缺点：干扰比较多，对温度和流速的变化比较敏感。适用范围：应用范围广，凡具氧化还原活性的或经衍生化后具氧化还原活性的物质。,优点：灵敏度高，选择性好，能同时给出组分的结构信息。缺点：响应信号受离子化效率限制，仪器较为昂贵，流动相必须是挥发性的，不能用非挥发性的缓冲盐及表面活性剂。适用范围：组分的结构鉴别，微量及痕量组分的分析，药物代谢分析等。,优点：通用型检测器，只要组分的折光率与流动相的折光率有足够的差别就能检测。缺点：灵敏度低、受环境温度、流量及流动相组成等波动的影响大，一般不能用于梯度洗脱。适用范围：RID为通用型检测器，适
18、用于无紫外吸收化合物的分析，如糖类分析。,2022/11/4,HPLC法在国内外药典中的应用与比较,23,检测器CHP(2010):优点：灵敏度高、噪音低、线性范围宽,USP and EP,2022/11/4,HPLC法在国内外药典中的应用与比较,24,USP and EPUSP(32)EP(6.0)Fixed,流动相,2022/11/4,HPLC法在国内外药典中的应用与比较,25,流动相CHP(2010)反相系统首选甲醇-水系统（采用紫外末,系统适用性System Suitability,2022/11/4,HPLC法在国内外药典中的应用与比较,26,系统适用性2022/10/9HPLC法在
19、国内外药典中的应用与,定义,CHP(2010):色谱系统的适用性试验通常包括理论板数、分离度、重复性和拖尾因子四个参数，其中，分离度和重复性尤为重要。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
20、integral system that can be evaluated as such.,2022/11/4,HPLC法在国内外药典中的应用与比较,27,定义CHP(2010):色谱系统的适用性试验通常包括理论板数,Definition,EP (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 o
21、f the equipment employed 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 chromat
22、ographic system are adequate for the analysis to be done.,2022/11/4,HPLC法在国内外药典中的应用与比较,28,DefinitionEP (6.0): The system,Definition,USP(32): No sample analysis is acceptable unless the requirements of system suitablity have been met. Sample analyses obtained while the system fails requirements are u
23、nacceptable. System suitability 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.,
24、2022/11/4,HPLC法在国内外药典中的应用与比较,29,DefinitionUSP(32): No sample,理论板数,评价色谱柱的重要指标。,影响因素：固定相、柱温、流动相和保留时间。*有争议时，以峰宽（W）计算结果为准,2022/11/4,HPLC法在国内外药典中的应用与比较,30,理论板数评价色谱柱的重要指标。CHP(2010) *USP(,分离度（R）,衡量色谱系统效能的关键指标！,2022/11/4,HPLC法在国内外药典中的应用与比较,31,分离度（R）衡量色谱系统效能的关键指标！ 2022/10/9,分离度（R）,* 有争议时，以峰宽（W）计算结果为准；# descr
25、ibed in individual monograph,2022/11/4,HPLC法在国内外药典中的应用与比较,32,分离度（R）CHP(2010)*R=2(tR2-tR1)/(,USP(32),Chromatographic 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
26、not less than 1.3; the resolution, R, between the famotidine and impurity D peaks is not less than 1.3;,Famotidine TabletsAssary,2022/11/4,HPLC法在国内外药典中的应用与比较,33,USP(32)Chromatographic system,EP(6.0),左甲状腺素钠含量测定：,左羟丙哌嗪对映体纯度：,2022/11/4,HPLC法在国内外药典中的应用与比较,34,EP(6.0)左甲状腺素钠含量测定：左羟丙哌嗪对映体纯度：2,重复性,评价连续进样中，色谱
27、系统响应值的重复性能。 CHP(2010):外标法：对照品溶液，连续进样5次，峰面积RSD不得过2.0%。内标法：配制相当于80%、100%、120%的对照品溶液，加入内标溶液，分别至少进样2次，计算平均校正因子的RSD不得过2.0% 。,2022/11/4,HPLC法在国内外药典中的应用与比较,35,重复性 评价连续进样中，色谱系统响应值的重复性能。202,重复性,USP(32):Unless otherwise specified in the individual monograph , data from five replicate injections of the analyte
28、 are used to calculate the relative standard deviation, SR, if the requirment is 2.0% or less; data from six replicate injections are used if the relative standard deviation requirment is more than 2.0%.,2022/11/4,HPLC法在国内外药典中的应用与比较,36,重复性 USP(32):2022/10/9HPLC法在国内,重复性,EP(6.0):The repeatability of r
29、esponse is expressed as an es timated percentage relative standard deviation(Sr(%) of a consecutive series of measurements for NOT fewer than 3 injections or applications of a reference solution.,mean of individual values,individual values expressed as peak area, peak height, or ratio of areas by th
30、e interal standardisation method,number of individual values,2022/11/4,HPLC法在国内外药典中的应用与比较,37,重复性 EP(6.0):mean of individu,重复性,EP(6.0):Unless otherwise prescribed, the maximum permitted RSD does not exceed the appropriate value given in table.,2022/11/4,HPLC法在国内外药典中的应用与比较,38,重复性 EP(6.0):Number of ind
31、ivi,重复性,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 freedom,EP(6.0): In an assay of
32、 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:,2022/11/4,HPLC法在国内外药典中的应用与比较,39,重复性NOTE: This requirement does,重复性,JP(XV): 公式与EP相同。重复次数及限度在各论中要求。例： Ritodrine Hydrochloride Relate
33、d 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%.,2022/11/4,HPLC法在国内外药典中的应用与比较,40,重复性 JP(XV): 公式与EP相同。2022/10/,
34、拖尾因子（对称因子）,用于评价色谱峰的对称性,2022/11/4,HPLC法在国内外药典中的应用与比较,41,拖尾因子（对称因子）用于评价色谱峰的对称性2022/10/9,拖尾因子T（or Symmetry factor）,2022/11/4,HPLC法在国内外药典中的应用与比较,42,拖尾因子T（or Symmetry factor）CHP(2,USP,It is also a common practice to measure the Asymmetry factor as the ratio of the distance between the vertical line conne
35、cting 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 the purpose of USP, only the formula presented in the Glossary of Symbols is valid.,2022/11/4,HPL
36、C法在国内外药典中的应用与比较,43,USPIt is also a common practic,EP中系统适用性：p/v and S/N,The peak-to-valley ratio (p/v) may be employed as a system suitability criterion in a test for related substances when baseline separation between 2 peaks is not achieved.p/v=Hp/Hv,For example: Econazole related substances,2022/1
37、1/4,HPLC法在国内外药典中的应用与比较,44,EP中系统适用性：p/v and S/NThe peak-t,EP,The short-term noise influences the precision of quantification.The signal-to-noise ratio is calculated using the following equation:S/N=2H/h,For example: ketotifen hydrogen fumarate related substances,2022/11/4,HPLC法在国内外药典中的应用与比较,45,EPThe
38、short-term noise influen,色谱条件的调整Adjustment of Chromatographic Conditions,2022/11/4,HPLC法在国内外药典中的应用与比较,46,色谱条件的调整2022/10/9HPLC法在国内外药典中的应,CHP(2010),明确规定：不可变的有：固定相的种类、流动相的组分、检测器类型可变的有：色谱柱内径、长度、载体粒度、流动相流速、混合流动相各组分的比例、柱温、进样量、检测器的灵敏度。,2022/11/4,HPLC法在国内外药典中的应用与比较,47,CHP(2010)明确规定：2022/10/9HPLC法在国,CHP(2010
39、),与CHP(2005)不同：中国药典2010年版规定了流动相调整的限度。调整流动相组分比例时，以组分比例较低者（50）相对于自身改变量不超过30且相对于总量的改变量不超过10为限，如30相对改变量的数值超过总量的10时，则改变量以总量的10为限。,2022/11/4,HPLC法在国内外药典中的应用与比较,48,CHP(2010)与CHP(2005)不同：中国药典20,CHP(2010),例：奥美拉唑肠溶片，释放度检查，色谱条件：,问题：峰型差！流动相可调范围：82.5:17.5 67.5:32.5解决：70：30,2022/11/4,HPLC法在国内外药典中的应用与比较,49,CHP(201
40、0)例：奥美拉唑肠溶片，释放度检查，色谱条件：,USP(32),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.,2022/11/4,HPLC法在国内外药典中的应用与比较,50,USP(32)pH of mobile phaseconc
41、e,USP(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 to 60:40. SPECIFIED RATIO OF 2:98 Thirty percen
42、t 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.,2022/11/4,HPLC法在国内外药典中的应用与比较,51,USP(32)Binary Mixtures 2022/,USP(32),Ternary Mixtures SPECIFIED RATIO OF 60:35:5 For the second component, 30% of 35 is 10.5% absolute, which exceeds the max
43、imum 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 component is used to give a total of 100%.Therefor
44、e, 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.,2022/11/4,HPLC法在国内外药典中的应用与比较,52,USP(32)Ternary Mixtures 2022,EP(6.0),The extent to which the various parameters of a chromatographic test may be adjusted to satisfy the system suitability criteria witho
45、ut fundamentally modifying the methods are listed below.Isocratic elution :,2022/11/4,HPLC法在国内外药典中的应用与比较,53,EP(6.0)The extent to which the,EP(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
46、 a minor component at 10 % of 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
47、% whereas a 2% absolute adjustment 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.,2022/11/4,HPLC法在国内外药典中的应用与比较,54,EP(6.0)Composition of the mobi,EP(6.0),When column dimensions are changed,the flow rate may be ajusted
48、as necessary using equation:,F1：规定的流速F2：调整后的流速l1：规定的柱长l2：实际所用的柱长d1：规定柱子的内径d2：实际所用柱子的内径,2022/11/4,HPLC法在国内外药典中的应用与比较,55,EP(6.0)When column dimensions,EP(6.0),Gradient elution：more critical than with isocratic elution,2022/11/4,HPLC法在国内外药典中的应用与比较,56,EP(6.0)pH of the aqueous conce,HPLC的应用及方法开发,2022/11/
49、4,HPLC法在国内外药典中的应用与比较,57,HPLC的应用及方法开发2022/10/9HPLC法在国内外,HPLC应用,HPLC 可以用来干什么？,2022/11/4,HPLC法在国内外药典中的应用与比较,58,HPLC应用类型定 义I原料药或药物制剂活性成分中主要成分,方法开发的指导原则,ICHUSP 中国药典,2022/11/4,HPLC法在国内外药典中的应用与比较,59,方法开发的指导原则ICH2022/10/9HPLC法在国内外,ICH,ICH(人用药品注册技术要求国际协调会)三方协调指导原则 Q2A：分析方法论证的文本ICH三方协调指导原则 Q2B：方法学ICH三方协调指导原则,
50、2022/11/4,HPLC法在国内外药典中的应用与比较,60,ICHICH(人用药品注册技术要求国际协调会)三方协调指导原,ICH,通常而非绝对 如已平评价重复性，可不再评价,2022/11/4,HPLC法在国内外药典中的应用与比较,61,ICH分析方法的类型鉴别杂质检查含量分析，溶出度，含量/效价,ICH,值得一提的：表中没有列出耐用性，但在分析方法研究过程的适当阶段，应予以考虑！如果测量结果对分析条件的变化时敏感的，那么该分析条件就应当适当控制或在方法中注明。通过耐用性评估，建立一系列的系统适用性参数，以确保在任何时候使用该分析方法都是有效的。在HPLC中典型变化的例子： 流动相pH值变
