1、HPLC法在國內(nèi)外藥典中的應(yīng)用與比較 山東省藥品檢驗所化學(xué)藥品科王小兵10/11/20221山東省藥品檢驗所匯報的主要內(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)10/11/20222山東省藥品檢驗所一、簡述中國藥典2010年版二部共收載2271個品種，新增品種330個，修訂品種1500個，涉及HPLC檢測項目的品種有1291個，占總品種的57%，其中新增/修訂926個。HPLC法在整個藥典品種的檢驗中占有重要地位。10/11/20223山東省藥品檢驗所High Performance Liquid Chromatography簡稱HPL

2、C，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：高壓+高速+高效+高靈敏度一、簡述10/11/20224山東省藥品檢驗所CHP(2010)：中國藥典2010年版USP：United Stated Pharmacopoeia EP：European PharmacopoeiaJP：Japanese Pharmacopoeia一、簡述10/11/20225山東省藥品檢驗所定義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山東省藥品檢驗所定義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山東省藥品檢驗所泵CHP(2010)USP(32)EP(6.0)

5、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）10/11/202210山東省藥品檢驗所進(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 / 自動強調(diào)重復(fù)性好即可10/11/202211山東省藥品檢驗所色譜柱分離的核心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

6、求：28；8，硅膠溶解以5m最為常見10/11/202212山東省藥品檢驗所色譜柱Zorbax StableBond柱：采用了較大的二異丁基(SB-C18)或二異丙基(SB-C8、SB-C3、SB-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)。10/11/202214山東省藥品檢驗所色譜柱Zorbax Extend C18柱：采用獨特的雙配位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。10/11/202215山東

7、省藥品檢驗所C-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）10/11/202217山東省藥品檢驗所L57L67未列出10/11/202218山東省藥品檢驗所EP(6.0) 2.2.29： 大部分分離機制都是基于以化學(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。10/11/202219山東省藥品檢驗所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山東省藥品檢驗所EPMannitol Assay:10/11/202221山東省藥品檢驗所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山東省藥品檢驗所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山東省藥品檢驗所流動相CHP(2010)反相系統(tǒng)首選甲醇-水系統(tǒng)（采用紫外末端波長檢測，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，有機相比例應(yīng)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山東省藥品檢驗所定義CHP(2010):色譜系統(tǒng)的適用性試驗通常包括理論板數(shù)、分離度、重復(fù)性和拖尾因子四個參數(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 ope

13、rations and samples to be analyzed constitute an integral system that can be evaluated as such. 10/11/202227山東省藥品檢驗所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山東省藥品檢驗所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山東省藥品檢驗所理論板數(shù)評價色譜柱的重要指標(biāo)。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、流動相和保留時間。*有爭議時，以峰寬（W）計算結(jié)果為準(zhǔn)10/11/202230山東省藥品檢驗所分離度（R）衡量色譜系統(tǒng)效能的關(guān)鍵指標(biāo)！ 10/11/202231山東省藥品檢驗所分離度（R）CHP(2010)*R=2(tR2-tR1)/(W1+W2)R=2(tR2-tR1)/1.70(W1,h/2+W2,h/2)除另有

18、規(guī)定外，應(yīng)大于1.5 USP(32) *# EP(6.0) # JP(XV)* 有爭議時，以峰寬（W）計算結(jié)果為準(zhǔn)；# described in individual monograph10/11/202232山東省藥品檢驗所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山東省藥品檢驗所EP(6.0)左甲狀腺素鈉含量測定：左羥丙哌嗪對映體純度：10/11/202234山東省藥品檢驗所重復(fù)性 評價連續(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

20、%、120%的對照品溶液，加入內(nèi)標(biāo)溶液，分別至少進(jìn)樣2次，計算平均校正因子的RSD不得過2.0% 。10/11/202235山東省藥品檢驗所重復(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 re

21、plicate injections are used if the relative standard deviation requirment is more than 2.0%. 10/11/202236山東省藥品檢驗所重復(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 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山東省藥品檢驗所重復(fù)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山東省藥品檢驗所重復(fù)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山東省藥品檢驗所重復(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 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山東省藥品檢驗所拖尾因子（對稱因子）用于評價色譜峰的對稱性10/11/202241山東省藥品檢驗所拖尾因子T（or Symmetry factor）CHP(2010)T=W0.05h/2d1應(yīng)符合個論項下的規(guī)定除另有規(guī)定外，峰高法定量時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 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山東省藥品檢驗所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山東省藥品檢驗所EP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 separation

30、 between 2 peaks is not achieved.p/v=Hp/HvFor example: Econazole related substances10/11/202244山東省藥品檢驗所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山東省藥品檢驗所色譜條件的調(diào)整Adjustment of Chromatographic Conditions10/11/202246山東省藥品檢驗所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、檢測器的靈敏度。10/11/202247山東省藥品檢驗所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，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

32、限，如30相對改變量的數(shù)值超過總量的10時，則改變量以總量的10為限。 10/11/202248山東省藥品檢驗所CHP(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)范圍：82.5:17.5 67.5:32.5解決：70：3010/11/202249山東省藥品檢驗所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山東省藥品檢驗所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山東省藥品檢驗所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山東省藥品檢驗所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山東省藥品檢驗所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山東省藥品檢驗所EP(6.0)When column dimensions are changed,the flow rate may be ajusted as necessary using equation:F1：規(guī)定的流速F2：調(diào)整后的流速l1：規(guī)定的柱長l2：實際所用的柱長d1：規(guī)定柱子的內(nèi)徑d2：實際所用柱子的內(nèi)徑10/11/202255山東省藥品檢驗所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山東省藥品檢驗所HPLC的應(yīng)用及方法開發(fā)10/11/202257山東省藥品檢驗所HPLC應(yīng)用類型定 義I

46、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)檢查或藥物制劑中降解產(chǎn)物的分析程序，這些分析程序包括定量試驗或限度試驗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HPLC 可以用來干什么？10/11/202258山東省藥品檢驗所方法開發(fā)的指導(dǎo)原則ICHUSP 中國藥典10/11/202259山東省藥品檢驗所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10/11/202260山東省藥品檢驗所ICH分析方法的類型項目鑒別雜質(zhì)檢查含量分析，溶出度，含量/效價定量限度檢測準(zhǔn)確度+精密度 重復(fù)性+中間精密度+ + 專屬性+檢測限度+定量限度+線性+范圍+通常而非絕對 如已平評價重復(fù)性，可不再評價10/11/202261山東省藥