hplc法在國內(nèi)外藥典中的應(yīng)用與比較解析

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藥品科王小兵,2024/3/12,山東省藥品檢驗所,2,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),2024/3/12,山東省藥品檢驗所,3,一、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

2、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。,2024/3/12,山東省藥品檢驗所,4,High Performance Liquid Chromatography簡稱HPLC，開始應(yīng)用于20世紀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,2024/3/12,山東省藥品檢驗所,5,C

3、HP(2010)：《中國藥典》2010年版USP：United Stated Pharmacopoeia EP：European PharmacopoeiaJP：Japanese Pharmacopoeia,一、簡述,2024/3/12,山東省藥品檢驗所,6,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，對供試品進行分離測定的色譜方法。USP(32)：High-pressure

4、 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, adsorp

5、tion, or ion-exchange processes, depending upon the type of stationary phase used.,2024/3/12,山東省藥品檢驗所,7,定義,EP(6.0) 2.2.29：Liquid chromatography is a method of chromatographic separation based on the difference in the dis

6、tribution of species between two non-miscible phases, in which percolates through a stationary phase contained in a column.,2024/3/12,山東省藥品檢驗所,8,定義,JP(XV)2.01：Liquid chromatography is a method to develop a mixture inject

7、ed 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 capac

8、ity against the stationary phase, and to determine the components.,2024/3/12,山東省藥品檢驗所,9,二、高效液相色譜儀,基本組成：,2024/3/12,山東省藥品檢驗所,10,泵,泵的種類很多，目前應(yīng)用最多的是柱塞往復泵（恒流泵）,2024/3/12,山東省藥品檢驗所,11,進樣器,2024/3/12,山東省藥品檢驗所,12,色譜柱—分離的核心,CHP(2010

9、)附錄：1.正相：硅膠柱； 反相： C-18柱、C-8柱；2.粒徑：普通3～10μm； < 2μm（亞-2μm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：2～8；,8，硅膠溶解,以5μm最為常見,2024/3/12,山東省藥品檢驗所,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)，給我們提供了更多的選擇。Gemini-NX C18柱：,色譜柱,Kinete

10、x C18柱：通過核-殼顆粒技術(shù)，使顆粒減小到2.6μm，而無需高壓即可達到提高速度、分離度和靈敏度的效果。,2024/3/12,山東省藥品檢驗所,14,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)鏈基團和空間位阻，避免了在低pH條件下的水解破壞，其pH1.0~8.0，溫度上限也可達到80~90℃，甚至在100%水相

11、中也有出色的表現(xiàn)。,2024/3/12,山東省藥品檢驗所,15,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pH2~11.5的范圍內(nèi)是穩(wěn)定的。,2024/3/12,山東省藥品檢驗所,16,USP(32):,Stationary phases for modern, reverse-phase liquid chromatography typica

12、lly consist of an organic phase chemically bound to silica or other materials.1. Particles：usually 3 to 10 μm in diameter；may range up to 50 μm or more for preparative columns. 2. Internal diameters：usually 2 to 5mm fo

13、r analytical separation，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:,C-1

14、8,硅膠柱,C-8,NH2柱,陽離子交換柱（SCX）,CN柱,苯基柱,陰離子交換柱（SAX）,L57~L67未列出,2024/3/12,山東省藥品檢驗所,19,EP(6.0) 2.2.29：,大部分分離機制都是基于以化學鍵合硅膠作為固定相，極性溶劑作為流動相的色譜條件。而化學鍵合相的性質(zhì)往往決定了色譜系統(tǒng)的分離性能。,2024/3/12,山東省藥品檢驗所,20,particle size：3~10μm internal diame

15、ters：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

16、(6.0) 2.2.29：,,2024/3/12,山東省藥品檢驗所,21,EP,Mannitol Assay:,,2024/3/12,山東省藥品檢驗所,22,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 us

17、ed.,2024/3/12,山東省藥品檢驗所,23,檢測器,CHP(2010):UVDADFLDECDELSDRIDMS,優(yōu)點：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：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：通用型檢測器，對各種物質(zhì)有幾乎相同的響應(yīng)。缺點：靈敏度相對較低，流動相必

18、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。,優(yōu)點：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：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

19、、多環(huán)芳烴、維生素、甾體化合物及酶等的檢測。,優(yōu)點：靈敏度很高，尤其適用于痕量組分分析。缺點：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：靈敏度高，選擇性好，能同時給出組分的結(jié)構(gòu)信息。缺點：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。,

20、優(yōu)點：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：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。,2024/3/12,山東省藥品檢驗所,24,USP and EP,2024/3/12,山東省藥品檢驗所,25,流動相,2024/3/12,山東省藥品檢驗所,26,,系統(tǒng)適用性System Suitabi

21、lity,2024/3/12,山東省藥品檢驗所,27,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、重復性和拖尾因子四個參數(shù)，其中，分離度和重復性尤為重要。ICH definition: System suitability testing is an integral part of many analytical procedures. The tests are based on the concept

22、 that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such.,2024/3/12,山東省藥品檢驗所,28,Definition,EP (6.0): The system suitability tests repr

23、esent an integral part of the method and are used to ensure adequate performance of the chromatographic system. The various components of the equipment employed must be qualified and be capable of achieving the precis

24、ion 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 chr

25、omatographic system are adequate for the analysis to be done.,2024/3/12,山東省藥品檢驗所,29,Definition,USP(32): No sample analysis is acceptable unless the requirements of system suitablity have been met. Sample analyses obtain

26、ed 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 sign

27、ificant change in equipment or in a critical reagent, suitability testing should be performed before the injection of samples.,2024/3/12,山東省藥品檢驗所,30,理論板數(shù),評價色譜柱的重要指標。,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,2024/3/12,山東省

28、藥品檢驗所,31,分離度（R）,衡量色譜系統(tǒng)效能的關(guān)鍵指標！,2024/3/12,山東省藥品檢驗所,32,分離度（R）,,* 有爭議時，以峰寬（W）計算結(jié)果為準；# described in individual monograph,2024/3/12,山東省藥品檢驗所,33,USP(32),Chromatographic system — The liquid chromatograph is equipped with a 27

29、5-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 pea

30、ks is not less than 1.3;,Famotidine TabletsAssary,2024/3/12,山東省藥品檢驗所,34,EP(6.0),左甲狀腺素鈉含量測定：,左羥丙哌嗪對映體純度：,2024/3/12,山東省藥品檢驗所,35,重復性,評價連續(xù)進樣中，色譜系統(tǒng)響應(yīng)值的重復性能。 CHP(2010):外標法：對照品溶液，連續(xù)進樣5次，峰面積RSD不得過2.0%。內(nèi)標法：配制相當于80%、100%、12

31、0%的對照品溶液，加入內(nèi)標溶液，分別至少進樣2次，計算平均校正因子的RSD不得過2.0% 。,2024/3/12,山東省藥品檢驗所,36,重復性,USP(32):Unless otherwise specified in the individual monograph , data from five replicate injections of the analyte are used to calculate the r

32、elative 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%.,2024/3/12,山東省藥品檢驗所,37,重復性,EP(6.0):The r

33、epeatability of response 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 o

34、f individual values,individual values expressed as peak area, peak height, or ratio of areas by the interal standardisation method,number of individual values,2024/3/12,山東省藥品檢驗所,38,重復性,EP(6.0):Unless otherwise prescribe

35、d, the maximum permitted RSD does not exceed the appropriate value given in table.,,2024/3/12,山東省藥品檢驗所,39,重復性,NOTE: This requirement does not apply to tests for related substances.,upper limit given in the definition of

36、the individual monograph minus 100%,contant(0.349),number of replicate injections of the reference solution (3≤n≤6),90% probability level，n-1 degrees of freedom,EP(6.0): In an assay of an active substance where the va

37、lue is 100 percent for a pure substance, the maximum permitted (Sr(%)max) for defined limits is calculated using the following equation:,2024/3/12,山東省藥品檢驗所,40,重復性,JP(XV): 公式與EP相同。重復次數(shù)及限度在各論中要求。例： Ritodrine Hydrochl

38、oride Related subsances System suitability— Test for required detectability: ............ System performance:............ System repeatability: When the test is repeated 6 times with ......，the relative st

39、andard deviation of the peak areas of deferoxamine is not more than 3.0%.,2024/3/12,山東省藥品檢驗所,41,拖尾因子（對稱因子）,用于評價色譜峰的對稱性,2024/3/12,山東省藥品檢驗所,42,拖尾因子T（or Symmetry factor）,2024/3/12,山東省藥品檢驗所,43,USP,It is also a common practic

40、e 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 me

41、asure 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.,2024/3/12,山東省藥品檢驗所,44,EP中系統(tǒng)適用性：p/v and S/N,The peak-to-va

42、lley 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,2024/3/12,

43、山東省藥品檢驗所,45,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

44、substances,2024/3/12,山東省藥品檢驗所,46,,色譜條件的調(diào)整Adjustment of Chromatographic Conditions,2024/3/12,山東省藥品檢驗所,47,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、進樣量、檢測器的靈敏度。,2024/3/12,山東省藥品檢驗所,4

45、8,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。,2024/3/12,山東省藥品檢驗所,49,CHP(2010),例：奧美拉唑腸溶片，釋放度檢查，色譜條件：,,問題

46、：峰型差！流動相可調(diào)范圍：82.5:17.5 ~ 67.5:32.5解決：70：30,2024/3/12,山東省藥品檢驗所,50,USP(32),If adjustments of operating conditions to meet system suitability requirments are necessary, each of the following is the maxium variation that c

47、an be considered, unless otherwise directed in the monograph.,,2024/3/12,山東省藥品檢驗所,51,USP(32),Binary Mixtures — SPECIFIED RATIO OF 50:50 —Thirty percent of 50 is 15% absolute, but this exceeds the maximum permitted chang

48、e 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% absolute. Therefore the maximu

49、m allowed adjustment is within the range of 1.4:98.6 to 2.6:97.4.,2024/3/12,山東省藥品檢驗所,52,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

50、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,

51、 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 61.5:35:3.5 would meet the requirement.,,2024/3/12,山東省藥品檢驗所,53,EP(6.0),The e

52、xtent 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.Isocratic elution :,,2024/3/12,山東省藥

53、品檢驗所,54,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 mobi

54、le 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% re

55、lative 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.,,2024/3/12,

56、山東省藥品檢驗所,55,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,,2024/3/12,山東省藥品檢驗所,56,EP(6.0),Gradient elu

57、tion：more critical than with isocratic elution,2024/3/12,山東省藥品檢驗所,57,,HPLC的應(yīng)用及方法開發(fā),2024/3/12,山東省藥品檢驗所,58,HPLC應(yīng)用,HPLC 可以用來干什么？,2024/3/12,山東省藥品檢驗所,59,方法開發(fā)的指導原則,ICHUSP 《中國藥典》,2024/3/12,山東省藥品檢驗所,60,ICH,ICH(人用藥品注冊技術(shù)要求國際協(xié)調(diào)會)

58、三方協(xié)調(diào)指導原則 Q2A：分析方法論證的文本ICH三方協(xié)調(diào)指導原則 Q2B：方法學ICH三方協(xié)調(diào)指導原則,2024/3/12,山東省藥品檢驗所,61,ICH,①通常而非絕對② △如已平評價重復性，可不再評價,2024/3/12,山東省藥品檢驗所,62,ICH,值得一提的：表中沒有列出耐用性，但在分析方法研究過程的適當階段，應(yīng)予以考慮！如果測量結(jié)果對分析條件的變化時敏感的，那么該分析條件就應(yīng)當適當控制或在方法中注明。通過耐用性

59、評估，建立一系列的系統(tǒng)適用性參數(shù)，以確保在任何時候使用該分析方法都是有效的。在HPLC中典型變化的例子： 流動相pH值變化 流動相組分變化 不同柱子 溫度 流速,2024/3/12,山東省藥品檢驗所,63,USP, Validation of compendial procedures,2024/3/12,山東省藥品檢驗所,64,USP,HPLC方法中的要求：,2024/3/

60、12,山東省藥品檢驗所,65,CHP(2010),《中國藥典》2010年版附錄ⅩⅨ A 藥品質(zhì)量標準分析方法驗證指導原則基本與ICH相同，增加了耐用性，且比ICH詳細。,2024/3/12,山東省藥品檢驗所,66,色譜柱的選擇,2024/3/12,山東省藥品檢驗所,67,色譜柱的選擇,特殊的：含光學異構(gòu)體（對映體）——特殊的手性柱含其他異構(gòu)體（立體、位置）——一般正相柱最佳，尤其用原硅膠柱無機鹽混合物——離子色譜法 ...

61、...,2024/3/12,山東省藥品檢驗所,68,耐用性,耐用性試驗經(jīng)常會提到用不同類型的色譜柱進行試驗。所謂的不同類型*：A型色譜柱：早期色譜柱，無定形或球形硅膠，硅膠純度較低，重金屬含量高，鍵合密度較低，碳載量較低，未采用封尾技術(shù)或不完全封尾技術(shù)，硅羥基活性高，殘留硅羥基導致的“第二效應(yīng)”較強，能與堿性化合物、酸性化合物發(fā)生氫鍵和離子交換作用，造成峰拖尾，但對于某些極性化合物，硅羥基是組分分離的主要因素。,*引自中檢所SOP,

62、2024/3/12,山東省藥品檢驗所,69,,B型色譜柱：球形高純硅膠，重金屬含量較低，鍵合密度較高，碳載量較高，采用非極性基團封尾技術(shù)，使硅羥基活性降低，能改善堿性化合物、酸性化合物的分離。E型色譜柱：2種亞型，Ec型采用極性基團封尾（如二醇基）， Eb型則是在十八烷基中嵌入極性基團（如甲酰胺基）使填料親水性增加，能用100%水溶液作為流動相，具有獨特選擇性。,2024/3/12,山東省藥品檢驗所,70,,不同的色譜柱公司都有不同的