1、HPLC法在国内外药典中法在国内外药典中的应用与比较的应用与比较2022-10-31HPLC法在国内外药典中的应用与比较汇报的主要内容 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2022-10-31HPLC法在国内外药典中的应用与比较一、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中占有重要地位。2022-10-31HPLC法在国内外药典中的应用与比较High Performance Li
2、quid 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2022-10-31HPLC法在国内外药典中的应用与比较CHP(2010)：中国药典2010年版USP：United Stated Pharmacopoeia EP：European PharmacopoeiaJP：Japanese Pharmacopoeia一、简述2022-10-31HPLC法在国内外药典中的应用与比较定义CHP(2010)附录V D：H
3、PLC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 chromatography,is a separation technique based on a solid stationary phase and a liquid mobile phase.Separations are achieved by partition,adsorption,or ion-exchan
4、ge processes,depending upon the type of stationary phase used.2022-10-31HPLC法在国内外药典中的应用与比较定义EP(6.0)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 ph
5、ase contained in a column.2022-10-31HPLC法在国内外药典中的应用与比较定义JP(XV)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
6、making use of the difference of retention capacity against the stationary phase,and to determine the components.2022-10-31HPLC法在国内外药典中的应用与比较二、高效液相色谱仪基本组成：2022-10-31HPLC法在国内外药典中的应用与比较泵CHP(2010)USP(32)EP(6.0)JP(XV)未解释满足等度和梯度洗脱;压力：5000psi或更高;最大可达10ml/min可满足等度或梯度洗脱只强调了恒定速率泵的种类很多，目前应用最多的是柱塞往复泵（恒流泵）2022-1
7、0-31HPLC法在国内外药典中的应用与比较进样器CHP(2010)USP(32)EP(6.0)JP(XV)未解释微量进样器定量环自动进样器手动/自动强调重复性好即可2022-10-31HPLC法在国内外药典中的应用与比较色谱柱分离的核心CHP(2010)附录：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2022-10-31HPLC法在国内外药典中的应用与比较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。Ge
8、mini-NX C18柱：色谱柱n乙烷交联，抵抗高pH侵蚀n多点配体键合，抵抗低pH下配体水解npH112稳定！uKinetex 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。2022-10-31HPLC法在国内外药典中的应用与比较色谱柱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。2022-10-3
9、1HPLC法在国内外药典中的应用与比较色谱柱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。2022-10-31HPLC法在国内外药典中的应用与比较USP(32):Stationary phases for modern,reverse-phase liquid chromatography typically consist of an organic phase chemically bound to silica or other materials.1.Particles：usu
10、ally 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Chromatographic reagents in the Reagents,Indicators,and Solutions secti
11、on列出了USP-NF所用到的不同类型的柱子，如下所示:2022-10-31HPLC法在国内外药典中的应用与比较C-18硅胶硅胶柱柱C-8NH2柱柱阳离子交换阳离子交换柱柱（SCX）CN柱柱苯基苯基柱柱阴离子交换阴离子交换柱柱（SAX）2022-10-31HPLC法在国内外药典中的应用与比较uL57L67未列出2022-10-31HPLC法在国内外药典中的应用与比较EP(6.0)2.2.29：u 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。2022-10-31HPLC法在国内外药典中的应用与比较 particle
12、 size：310m internal diameters：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：ustationary phase degradetionucomposition of the mob
13、ile phase2022-10-31HPLC法在国内外药典中的应用与比较EPMannitol Assay:2022-10-31HPLC法在国内外药典中的应用与比较JP(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.2022-10-31HPLC法在国内外药典中的应用与比较检测器CHP(2010):UVDADFLDECDELSDRIDMSu优点：优点：灵敏度高、噪音
14、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。u 缺点：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，流动相的截止波长必须小于检测波长。u 适用范围：适用范围：大多数有紫外吸收的化合物。u优点：通用型检测器，对各种物质有几乎相同的响应。u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。u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。u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。u 缺点：只适用于能够产生
15、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。u 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、甾体化合物及酶等的检测。u优点：灵敏度很高，尤其适用于痕量组分分析。u缺点：干扰比较多，对温度和流速的变化比较敏感。u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。u优点：灵敏度高，选择性好，能同时给出组分的结构信息。u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。u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。u优点：通用型检测器，
16、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。u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。u适用范围：RID为通用型检测器，适用于无紫外吸收化合物的分析，如糖类分析。2022-10-31HPLC法在国内外药典中的应用与比较USP and EPUSP(32)EP(6.0)Fixed,variable and multi-wavelength detectors are widely available.UV、RID、FLD、ECD.New detectors continue to be developed in attempts to overc
17、ome the deficiencies of those being used.UV/Vis spectrophotometers,including diode array detectors,are the most commonly employed detectors.2022-10-31HPLC法在国内外药典中的应用与比较流动相CHP(2010)反相系统首选甲醇-水系统（采用紫外末端波长检测，首选乙腈-水系统）缓冲盐：少用，尽可能低反相色谱系统，C-18柱，有机相比例应不低于5%。USP(32)High-purity reagents and“HPLC grade”organic
18、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 helium,sonication or using on-line membrane/vacuumAdjustment of the pH,is effected using only the aqueous compone
19、nt of the mobile phase and not the mixture.2022-10-31HPLC法在国内外药典中的应用与比较系统适用性System Suitability2022-10-31HPLC法在国内外药典中的应用与比较定义 CHP(2010):色谱系统的适用性试验通常包括理论板数理论板数、分离度分离度、重复性重复性和拖尾因子拖尾因子四个参数，其中，分离度和重分离度和重复性复性尤为重要。ICH definition:System suitability testing is an integral part of many analytical procedures.T
20、he tests are based on the concept that the equipment,electronics,analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such.2022-10-31HPLC法在国内外药典中的应用与比较Definition EP(6.0):The system suitability tests represent an integral part of the method and are u
21、sed to ensure adequate performance of the chromatographic system.The various components of 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
22、methods.They are used to verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis to be done.2022-10-31HPLC法在国内外药典中的应用与比较Definition USP(32):No sample analysis is acceptable unless the requirements of system suitablity have been met.Sample analyses ob
23、tained while the system fails requirements are unacceptable.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 b
24、e performed before the injection of samples.2022-10-31HPLC法在国内外药典中的应用与比较理论板数评价色谱柱的重要指标。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：半高峰宽N=5.54(tR/W0.5h)2W0.5h：半高峰宽u 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2022-10-31HPLC法在国内外药典中的应用与比较分离度（R）u衡量色谱系
25、统效能的关键指标！2022-10-31HPLC法在国内外药典中的应用与比较分离度（R）CHP(2010)*R=2(tR2-tR1)/(W1+W2)R=2(tR2-tR1)/1.70(W1,h/2+W2,h/2)除另有规定外，应大于1.5 USP(32)*#EP(6.0)#JP(XV)*有争议时，以峰宽（W）计算结果为准；#described in individual monograph2022-10-31HPLC法在国内外药典中的应用与比较USP(32)Chromatographic system The liquid chromatograph is equipped with a 275
26、-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;Famotidine TabletsAssary 2022-10-31HPLC法在国内外药典中的应用与比较EP(6.0)u左甲状腺素钠含量测定：
27、u左羟丙哌嗪对映体纯度：2022-10-31HPLC法在国内外药典中的应用与比较重复性 评价连续进样中，色谱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%。2022-10-31HPLC法在国内外药典中的应用与比较重复性 USP(32):Unless otherwise specified in the individual monograph,data from five replicate injection
28、s of the analyte 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%.2022-10-31HPLC法在国内外药典中的应用与比较重复性 EP(6.0):The repeatability of response is expressed as
29、 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 valuesindividual values expressed as peak area,peak height,or ratio of areas by the interal standardisation me
30、thodnumber of individual values2022-10-31HPLC法在国内外药典中的应用与比较重复性 EP(6.0):uUnless 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
31、.921.063.00.620.891.101.272022-10-31HPLC法在国内外药典中的应用与比较重复性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
32、degrees of freedomEP(6.0):u In an assay 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:2022-10-31HPLC法在国内外药典中的应用与比较重复性 JP(XV):公式与EP相同。重复次数及限度在各论中要求。例：Ritodrine Hydrochloride Relat
33、ed 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%.2022-10-31HPLC法在国内外药典中的应用与比较拖尾因子（对称因子）用于评价色谱峰的对称性2022-10-31HPLC法在国
34、内外药典中的应用与比较拖尾因子T（or Symmetry factor）CHP(2010)T=W0.05h/2d1 应符合个论项下的规定 除另有规定外，峰高法定量时T应在0.951.05之间USP(32)T=W0.05/2fEP(6.0)As=W0.05h/2d An As value of 1.0 signifies symmetry.When As 1.0,the peak is tailing.When As 1.0,the peak is fronting.In a related substances test or assay,for a peak in the chromatog
35、ram 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/2f2022-10-31HPLC法在国内外药典中的应用与比较USPIt is also a common practice to measure the Asymmetry factor as the ratio of the distance between the vertical line connecting
36、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.2022-10-31HPLC法在国内外药典中的应用与
37、比较EP中系统适用性：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 separation between 2 peaks is not achieved.p/v=Hp/HvuFor example:Econazole related substances2022-10-31HPLC法在国内外药典中的应用与比较EPThe short-term noise influenc
38、es the precision of quantification.The signal-to-noise ratio is calculated using the following equation:S/N=2H/hu For example:ketotifen hydrogen fumarate related substances2022-10-31HPLC法在国内外药典中的应用与比较色谱条件的调整Adjustment of Chromatographic Conditions2022-10-31HPLC法在国内外药典中的应用与比较CHP(2010)明确规定：明确规定：不可变的有不
39、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。2022-10-31HPLC法在国内外药典中的应用与比较CHP(2010)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。2022-10-31HPLC法在国内外药典中的应用与比较CHP(2010)例：奥美拉唑肠溶片，释放度检查，色谱条件：u问题
40、：峰型差！u流动相可调范围：82.5:17.5 67.5:32.5u解决：70：302022-10-31HPLC法在国内外药典中的应用与比较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 within 10%provided the p
41、ermitted 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 diameterparticle sizeflow rat
42、ecolumn temperature70%25%can be reduced by as much as 50%50%10u 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.2022-10-31HPLC法在国内外药典中的应用与比较USP(
43、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 percent of 2 is 0.6
44、%absolute.Therefore the maximum allowed adjustment is within the range of 1.4:98.6 to 2.6:97.4.2022-10-31HPLC法在国内外药典中的应用与比较USP(32)Ternary Mixtures SPECIFIED RATIO OF 60:35:5 For the second component,30%of 35 is 10.5%absolute,which exceeds the maximum permitted change of 10%absolute in any component.
45、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%.Therefore,mixture ranges of 50:45:5 to 70:25:5 or 58.5:35:6.5 to 61.5:35
46、:3.5 would meet the requirement.2022-10-31HPLC法在国内外药典中的应用与比较EP(6.0)uThe extent to which the various parameters of a chromatographic test may be adjusted to satisfy the system suitability criteria without fundamentally modifying the methods are listed below.uIsocratic elution:pH of the aqueous compon
47、ent of the mobile phaseconcentration of salts in buffercomposition of the mobile phaseWavelengthcolumn temperature0.2pH 1.0pH(non-ionisable)within 10%Minor component 30%relative or 2%absolute whichever is larger.No component can be altered by more than 10%absolute（see example）no adjustment permitted
48、10column lengthinteral diameterparticle sizeflow rateinjection volume70%25%maximum reduction of 50%no increase permitted50%a larger adjustment is acceptable（see example）may be decreasedno increase permitted2022-10-31HPLC法在国内外药典中的应用与比较EP(6.0)Composition of the mobile phase:the amount of the minor sol
49、vent component may be adjusted by 30%relative or 2%absolute,whichever is the larger;for 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
50、 at 5%of the mobile phase,a 30%relative adjustment allows a range of 3.5-6.5%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.2022-10-31HPLC法在国内外药典中的应用与比较EP(6.0)When column dimensions are ch
