HPLC法在国内外药典中的应用与比较课件

1、HPLC法在国内外药典中的应用与比较 山东省药品检验所化学药品科王小兵10/11/20221山东省药品检验所汇报的主要内容 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10/11/20222山东省药品检验所一、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。10/11/20223山东省药品检验所High Performance Liquid Chromatography简称HPL

2、C，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10/11/20224山东省药品检验所CHP(2010)：中国药典2010年版USP：United Stated Pharmacopoeia EP：European PharmacopoeiaJP：Japanese Pharmacopoeia一、简述10/11/20225山东省药品检验所定义EP(6.0) 2.2.29：Liquid chromatography is a method of chroma

3、tographic 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.10/11/20227山东省药品检验所定义JP(XV)2.01：Liquid chromatography is a method to develop a mixture injected into a column prepared wit

4、h 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 determine the components.10/11/20228山东省药品检验所泵CHP(2010)USP(32)EP(6.0)

5、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）10/11/202210山东省药品检验所进样器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10/11/202211山东省药品检验所色谱柱分离的核心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

6、求：28；8，硅胶溶解以5m最为常见10/11/202212山东省药品检验所色谱柱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。10/11/202214山东省药品检验所色谱柱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。10/11/202215山东

7、省药品检验所C-18硅胶柱C-8NH2柱阳离子交换柱（SCX）CN柱苯基柱阴离子交换柱（SAX）10/11/202217山东省药品检验所L57L67未列出10/11/202218山东省药品检验所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。10/11/202219山东省药品检验所particle size：310m internal diameters：prescribed in the monograph, eg:pH：silica based reversed-phase col

8、umns 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 phase10/11/202220山东省药品检验所EPMannitol Assay:10/11/202221山东省药品检验所JP(XV)：A column with a statio

9、nary phase chemically bound on the inside wall instead of the column packed with the packing material may be used.10/11/202222山东省药品检验所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

10、ome the deficiencies of those being used.UV/Vis spectrophotometers, including diode array detectors, are the most commonly employed detectors.10/11/202224山东省药品检验所流动相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 solve

11、ntswater: 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 component

12、 of the mobile phase and not the mixture.10/11/202225山东省药品检验所定义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 ope

13、rations and samples to be analyzed constitute an integral system that can be evaluated as such. 10/11/202227山东省药品检验所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 compone

14、nts 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 methods. They are used to verify that the resolution and reproducibility of the ch

15、romatographic system are adequate for the analysis to be done.10/11/202228山东省药品检验所DefinitionUSP(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 suitability must be

16、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.10/11/202229山东省药品检验所理论板数评价色谱柱的重要指标。CHP(20

17、10) *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 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10/11/202230山东省药品检验所分离度（R）衡量色谱系统效能的关键指标！ 10/11/202231山东省药品检验所分离度（R）CHP(2010)*R=2(tR2-tR1)/(W1+W2)R=2(tR2-tR1)/1.70(W1,h/2+W2,h/2)除另有

18、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 monograph10/11/202232山东省药品检验所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

19、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 10/11/202233山东省药品检验所EP(6.0)左甲状腺素钠含量测定：左羟丙哌嗪对映体纯度：10/11/202234山东省药品检验所重复性 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

20、%、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% 。10/11/202235山东省药品检验所重复性 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 re

21、plicate injections are used if the relative standard deviation requirment is more than 2.0%. 10/11/202236山东省药品检验所重复性 EP(6.0):The repeatability of response is expressed as an es timated percentage relative standard deviation(Sr(%) of a consecutive series of measurements for NOT fewer than 3 injection

22、s 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 values10/11/202237山东省药品检验所重复性 EP(6.0):Unless otherwise prescribed, the maximum permitted RSD does not

23、exceed the appropriate value given in table.Number of individual injections3456B(percent)Maximum permitted relative standard deviation2.00.410.590.730.852.50.520.740.921.063.00.620.891.101.2710/11/202238山东省药品检验所重复性NOTE: This requirement does not apply to tests for related substances.upper limit give

24、n 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 assay of an active substance where the value is 100 percent for a pure substance, the maximum permitted (Sr

25、(%)max) for defined limits is calculated using the following equation:10/11/202239山东省药品检验所重复性 JP(XV): 公式与EP相同。重复次数及限度在各论中要求。例： Ritodrine Hydrochloride Related subsances System suitability Test for required detectability: . System performance:. System repeatability: When the test is repeated 6 times

26、with .，the relative standard deviation of the peak areas of deferoxamine is not more than 3.0%.10/11/202240山东省药品检验所拖尾因子（对称因子）用于评价色谱峰的对称性10/11/202241山东省药品检验所拖尾因子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/2dAn As value of 1.0 signifi

27、es 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 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/2f10/11/202

28、242山东省药品检验所USPIt is also a common practice 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 wou

29、ld be (W0.10-f0.10)/f0.10However, for the purpose of USP, only the formula presented in the Glossary of Symbols is valid. 10/11/202243山东省药品检验所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

30、 between 2 peaks is not achieved.p/v=Hp/HvFor example: Econazole related substances10/11/202244山东省药品检验所EPThe 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 subs

31、tances10/11/202245山东省药品检验所色谱条件的调整Adjustment of Chromatographic Conditions10/11/202246山东省药品检验所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。10/11/202247山东省药品检验所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

32、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。 10/11/202248山东省药品检验所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：70：3010/11/202249山东省药品检验所USP(32)pH of mobile phaseconcentration of saltsin bufferratio of components in mobile phaseWavelength of UV detectorinjection volumepH of the aqueous bu

33、ffer within 0.2 units of the value or range specified within 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 preci

34、sion and detection limitscolumn lengthcolumn inner diameterparticle 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 consid

35、ered, unless otherwise directed in the monograph.10/11/202250山东省药品检验所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

36、the range of 40:60 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. 10/11/202251山东省药品检验所USP(32)Ternary Mixtures SPECIFIED RATIO OF 60:35:5 For the second component, 30% of 35 is 10.5% absolut

37、e, 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 component is used to give

38、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. 10/11/202252山东省药品检验所EP(6.0)The extent to which the various parameters of a chromatographic test may be adjusted to satisfy the system suitability criteria without fundamentally m

39、odifying 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 component 30% relative or 2% absolute whichever is larger.No

40、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 acceptable（see example）may be decreasedno increase permitted10/11/

41、202253山东省药品检验所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 a minor component at 10 % of the mobile phase, a 30 % relative adjustment allows a range of 7-13% wherea 2% absolute adjustme

42、nt 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 adjustment allows a range of 3-7%, the absolute value being in this case the larger.No other component

43、 is altered by more than 10% absolute.10/11/202254山东省药品检验所EP(6.0)When column dimensions are changed,the flow rate may be ajusted 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10/11/202255山东省药品检验所EP(6.0)pH of the aqueous component of the mobile phaseconcentratio

44、n of salts in buffercomposition of the mobile phaseWavelength of UV detectorcolumn temperatureno adjustmentpermitted no adjustment permittedthe system suitability requirements are fulfilleldthe principal peak elute within 15% of the indicated retention timeelution power is not weaker than beforeno a

45、djustment permitted5column lengthinteral diameterparticle sizeflow rateinjection volume70%25%no adjustment permittedsame asisocratic elution may be decreasedno increase permittedGradient elution：more critical than with isocratic elution10/11/202256山东省药品检验所HPLC的应用及方法开发10/11/202257山东省药品检验所HPLC应用类型定 义I

46、原料药或药物制剂活性成分中主要成分的分析程序-如含量测定II原料药中杂质检查或药物制剂中降解产物的分析程序，这些分析程序包括定量试验或限度试验III用于性能参数测试的分析程序-如溶出度、释放度IV鉴别试验HPLC 可以用来干什么？10/11/202258山东省药品检验所方法开发的指导原则ICHUSP 中国药典10/11/202259山东省药品检验所ICHICH(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10/11/202260山东省药品检验所ICH分析方法的类型项目鉴别杂质检查含量分析，溶出度，含量/效价定量限度检测准确度+精密度 重复性+中间精密度+ + 专属性+检测限度+定量限度+线性+范围+通常而非绝对 如已平评价重复性，可不再评价10/11/202261山东省药