1、動植物、微生物來源的化學藥品質量控制中生物測定法與理化測定法的合理應用Reasonably apply biological and physical-chemical assay and test for the drugs, which extracted from animal and plant and microbes河北省藥品檢驗所楊梁Hebei provincial Institute for drug control Yang Liang1摘 要ABSTRACT 科學合理地應用生物測定法、理化測定法，制訂能夠有效控制藥品質量的藥品標準，不斷提高藥品的有效性和安全性是藥品標準工作

2、永恒的目標。 Work out drug standard ， that is able to control quality of drugs effectively, to improve safety and effectiveness of drug substances and drug product, is eternal goal for drug standard work.2 控制藥品質量的實質就是要控制原料藥和制劑中的藥物活性成分分子在其有效期內符合臨床需要的藥物構效關系并保持其物理穩定性、化學穩定性、治療學穩定性、微生物學穩定性和毒理學穩定性。 Control mole

3、cular of activity pharmaceutical ingredient in the drug substances and products, in essence of control quality， conform to structure-activity relationship and maintain its physical stability, chemical stability, therapeutic stability, microbiological stability and toxicology stability, before term o

4、f validity. 3 因此，我們認為在方法學研究中應該注意以下3點： 1、生物測定法是不可替代的方法。 2、理化測定法檢測結果必須體現藥物的真實活性或效價。 3、科學合理地綜合運用生物測定法和理化測定法，才能有效控制藥品質量。4 Therefore we must be attention to following 3 problems，when study and design for methodology: 1. Biological assay and test is the irreplaceable method. 2. Determination results of ph

5、ysical and chemical assay must be indicating true activity or potency of drug. 3. Only scientifically and reasonably apply biological and physical and chemical test methods, drug quality is controlled effectively.5一、生物測定法是無可替代的藥品檢測方法。 Biological assay is the irreplaceable method of drug test. 藥物的分子結

6、構及其構型、構象決定著藥物的生物學特性和理化特性，（特別是藥物制劑中藥物分子的結構是否穩定？其構型、構象對于治療或預防疾病是否有利？其降解產物、可能的外來物質會對人體產生何種危害？），而檢測與6 評價其特性所導致的生物效應（包括對疾病的治療作用與可能發生的不良反應）最直接的方法就是生物測定法。 The molecular structure of API in the drug product and its configuration and conformation decided its biological, physical and chemical characteristics,

7、 (especially must be attention for that, the structure and its configuration 7 and conformation whether or not stabilize？whether or not advantageous for the resist diseases or prevent diseases？its degradation substances and foreign material how hazard for human body？) Biological assay and test are m

8、ost direct methods, which can test and evaluate biological effect (including therapeutic action and undesirable action). 8 1.生物測定法是直接體現真實治療作用的方法 Biological assay is the method of directly reflecting a real cure action of drug product. 縮宮素源自豬牛羊垂體后葉的提取物。是臨床用于引產、產前子宮收縮不良和產后及子宮肌瘤術后的止血藥，其低劑量可增強子宮節律性收縮、高劑

9、量則可使肌層內血管受壓而止血。因而至今在臨床廣泛應用。9 檢測時測定的子宮肌肉收縮的高度與縮宮素的效價單位數相關顯著。所以其大鼠離體子宮測定法直接代表了臨床療效。 Oxytocin is extracted from hypophysinum of pig, cattle and sheep or goat. Oxytocin is treating for induction of labor, improve uterus contracts and postpartum styptic or after resection of uterus myoma, its lower dose

10、may impel the uterus contract, and its higher dose may control postpartum uterine bleeding by pressing vessel in muscle and therefore oxytocin is applying widely in hospitals.10 The potency of oxytocin is estimated by comparing the contraction effect produced on isolated rat uterus with that produce

11、d by Posterior Pituitary or Oxytocin Standard. It reflects the effect of clinic. The contraction of uterus has the remarkable correlation with the potency of oxytocin.11圖1：縮宮素使子宮收縮高度發生變化的記錄比較圖Figure 1：comparing the contraction effect produced on isolated rat uterus with that produced by Posterior Pi

12、tuitary or Oxytocin Standard.12表1：縮宮素使子宮收縮高度發生變化的數據表Table 1：date comparing the contraction effect produced on isolated rat uterus with that produced by Posterior Pituitary or Oxytocin Standard.13表2：縮宮素效價測定的生物統計和可靠性測驗結果Table 2：biological statistical results of the contraction effect produced on isola

13、ted rat uterus with that produced by Posterior Pituitary with Oxytocin standard potency.14 2、生物測定法是直接代表藥品安全性的有效方法 Biological assay is the effective method that directly indicated the drug safety. 洋地黃毒苷是一個源自紫花洋地黃的強心苷，其有效量為0.05至0.1mg，其極量為1.2mg，即有效量和中毒量之間距離很小，用鴿法進行生物測定，可以直接有效地測定其效價和中毒劑量，從而有效控制其制劑含量，為臨床

14、治療慢性心衰并防止洋地黃中毒，提供安全保證。15 Digitoxin is a kind of cardiac glycoside come from Digitalis purpurea L. Its effective dose is from 0.05 to 0.1mg. Its maximum dose is 1. 2mg, The distance is so small from effective dose to poisoning dose. The potency of digitalis may be determined by comparing the minimum

15、lethal dose it produced by pigeons with that produced by Digitalis Standard. This method can be used to effectively control the contents of a preparation of digitalis and then give a safety to be a treatment to heart failure and avoid digitalis poison.16 3、對于硫酸慶大霉素等多組分抗生素，由于其各個組分HPLC法測定法的響應值與其效價的量效關

16、系尚不明確，所以在用理化測定法控制其組分比例的同時，還必須用微生物測定法測定各組分的總效價，以控制其總體活性。17 Because relationship of these peaks responding by HPLC of every constitute with its dose- effect relationship of potency is not explicitness in order to control biological activity, therefore must be determinate total potency by microbiologic

17、al assay of antibiotics, simultaneously determinate proportion of constitutes by physical-chemical assay, for example gentamicin sulfate etc, of multi- constitute antibiotics.18表3：硫酸慶大霉素各組分和效價測定結果表Table 3：assay results of ratio of gentamicin components and total potency.19二、理化測定法的檢測結果必須真實體現被測物的生物活性和

18、安全性 The test results of physical and chemical determination must be truly embodied the biological activity and safety of the sample 1.為了保護環境，保護動物，我們應當努力研究取代生物測定法的理化分析法。 In order to protect the environment and animals, we should study physical and chemical analysis methods hard to instead of biologic

19、al assays. 20 2.采用的理化測定法必須用生物測定法驗證與評價，即理化測定法檢測目標物及其測定結果必須能體現其生物活性。 Analytical target components and determinable result by the physical and chemical determination method must be able to verify and evaluated by biological assays, namely the results of physical and chemical determination must be able

20、to embody the biological activity.21 FB0601是一個-內酰胺類半合成抗生素，其申報標準中的含量測定方法為中和滴定法。我們以金黃色葡萄球菌CMCC（B)26003為檢定菌，pH6.5II號培養基和pH6.0緩沖液，用微生物檢定法測定了其效價，結果表明：不同生產廠家樣品的中和滴定法測定結果與測得效價之間差異顯著。22 FB0601 is a half- synthetic -lactam antibiotic, using neutralization titration to determine the content in its application

21、 registered standard. With Staphyl. aureus CMCC(B) 26003 as test organism， pH6.5 II as medium ，pH6.0 sterile buffer， its titer evaluation by microbiological assay of antibiotics . The results show that ：significant difference between titer evaluation and titration coming from different manufacturers

22、 samples. 23 繼而，我們采用ODS柱，以磷酸鹽緩沖液：乙腈（75：25）為流動相，流速每分鐘1ml,紫外檢測波長225nm的HPLC法檢測，結果發現在8批不同工藝的產品中，既有單個主要色譜峰(A)者，也有兩個主要色譜峰(A、B)者。凡僅有單個色譜峰A者，其效價均遠高于有兩個色譜峰A、B者，（見下圖）。24 Then ,we adopt HPLC,ODS column, phosphate buffer: acetonitrile (75:25) as mobile phase, flow rate：1ml/min, and determination wavelength 225n

23、m by UV detector .We found both single principal peak (A) and two principal peaks (A and B) in eight production technology. Only single principal peak (A), its potency are far than two principal peaks of A and B (see chart as below).25圖2：FB0601的HPLC圖。上圖為僅有單一組分峰，下圖為有兩個主組分峰。Figure2：determined results

24、of FB0601 by HPLC .top: single principal peak (A); below: two principal peaks (A and B).min010203040mAU 0500100015002000 VWD1 A, Wavelength=225 nm (FBXL0314YP06.D) 4.910 9.345 10.735 12.303 21.430 A 36.827min010203040mAU 025050075010001250150017502000 VWD1 A, Wavelength=225 nm (FBXL0314YP05.D) 3.894

25、 4.804 7.193 9.050 9.957 C 11.863 18.057 B 20.465 A 21.612 23.365 34.749 41.55026 顯而易見，該HPLC法的專屬性和與體外抗菌活性的相關性均高于中和法。 Obviously, the method of HPLC is better than titration method in specificity and correlativity of antimicrobial action in vitro.27三、科學合理地綜合運用生物測定法和理化測定法Use reasonably and compositely

26、the biological, physical, chemical assay and test 為了既保護環境、節約資源，又確保藥品標準能對藥品的有效性、安全性和質量穩定性（包括物理、化學、治療學、微生物學和毒理學穩定性）實施有效控制，我們認為：在制訂或修訂藥品標準時，必須科學合理地綜合運用生物測定法和理化測定法。28 The author think: when we work out drug standard, we may use reasonably and compositely the biological testing methods and physical-chemi

27、cal testing methods to make the validity, safety, and stability of drug quality for sure and to achieve the goal of protecting environment and resource economy at the same time.29 1、不斷改進理化測定法的專屬性并采用生物測定法對其驗證與評價。 Improve continuously the specificity of physical-chemical method and use biological meth

28、od to verify and evaluate （1）方法學研究的專屬性不但是指可能存在的某組分（如雜質、降解物、基質等）時，對被分析物準確可靠測定的能力，還應指藥物活性成分的分子必須是具有可靠臨床療效的特定構型、構象（包括多晶和異構）和足夠的純度，并具備經生物測定法實驗確證的控制能力。30 The specificity of methodology research is not only means the ability to assess unequivocally the analysis in the presence of components which may be e

29、xpected to be present, (typically these might include impurities, degraded materials, matrix, etc.), accurately dependably the analyzed substance, but also the ability to control the molecule of active pharmaceutical ingredient to have special configuration and conformation that includes polymorph a

30、nd isomer of molecule, and enough purity. At the same time the method should have definite proof by biological method.31 （2）到目前為止，藥物的構效關系都是用單晶X射線衍射法與生物測定法結合而確定的。因此，結合臨床治療學效果、已有的構效關系和生物測定法對其進行驗證與評價，可為分析方法學研究提供選擇和改進的依據。32 Current medicines structure-activity relationship is determined by single crysta

31、l X-ray diffraction and biological method, so combining clinic therapeutic effect with the existing structure-activity relationship and biological method to verify and evaluate it may supply a choosing and improving foundation for analysis research.33 （3）從對于藥品的有效性、安全性和穩定性等都能夠實施有效控制的目標來說，沒有一種方法可以同時識別

32、藥物的生物及理化特性，換言之，沒有一種方法是萬能的。所以，只能綜合應用生物測定法和理化測定法。34 There is no all-powerful testing method to identify biological and physical-chemical characteristic of drug at the same time to the goal of controlling drug substances and drug products efficacy, safety, and stability. Wherefore we should do is to co

33、mbine the biological testing method and physical-chemical testing method.35 2、加強對生產過程中可能產生的有關物質包括原料和輔料的同系物、降解物、異構體等和外來物質（包括包裝材料及提取、精制過程中混入的雜質等），尤其要格外關注HPLC法之紫外檢測器不能識別的有關物質的控制和預防。36 We also need strengthen to reinforce the control possible relative materials include raw materials, homologous compound

34、 of accessories and degraded-materials, isomer and foreign material etc. ,(that includes parking materials and impurity from extracting and refining processes), especially pay attention to relative material that cant be identified by ultraviolet detector etc. of HPLC.37 3、為了保護環境，節約資源和減少污染，應按真實、準確、安全

35、、可靠與精簡高效的方針，綜合運用生物測定和理化測定法設計和制定藥品標準。 For protecting environment, resource economy and decrease polluter according to guideline principle, which conform to true, accurate, safety, confidence, simplify and efficient, guiding principle, we may reasonably apply biological and physical and chemical assay

36、 and test, to design drug standards.38 （1）鑒別 Identification IR法 Infrared spectroscopy 鹽酸林可霉素具有多晶現象。兩種晶型的紅外光譜行為差異顯著，為此，我們進行了晶體結構研究。 Lincomycin hydrochloride is having monograph. Its pattern of two crystal forms are different on middle infrared spectrum, because we study crystal structure of its two c

37、rystal forms.39 a晶型I. b.晶型a. crystal form b. crystal form圖3鹽酸林可霉素兩種的掃描電鏡照片Figure 3： photographs of Crystal polymorphs of lincomycin hydrochloride by scan electron microscopy.40 單晶射線衍射測定結果 Result by single crystal -ray diffraction 其晶型I屬正交晶系的P22121空間群晶胞參數為a=6.1670(31)，b=18.5350(8) ，c=20.6170(5) ，每個晶胞內

38、含有4個鹽酸林可霉素分子與4個水分子，化學計量式為C18H34N2O6SHCLH2O，計算分子量為461.01。41 林可霉素分子五元環（古液酸）為信封式構象，六元環（林可霉糖）為椅式構象，1位疏甲基，4位羥甲基為直立鍵，2、3位羥基及5位取代基均為平伏鍵。其分子內氫鍵為O（4）O(2);2.8295；分子間氫鍵為O（1）O（3）（1+x,y,z）:2.9235，水分子與林可霉素分子間氫鍵：O（w1）O（2）(-2-x,-1/2-y,-1/2+z):2.8849。晶態下分子以氫鍵力和范德華力維持空間穩定排列，見下圖。42 The crystal form I belongs orthorhom

39、bic system, in space group P22121, Z=4, with unit cell parameters a=6.1670(31) ，b=18.5350(8) ，c=20.6170(5) .stoichiometric formula: C18H34N2O6SHCLH2O，calculated molecular weight: 461.01.43圖4鹽酸林可霉素晶型分子立體結構投影圖Figure 4： stereo-structure perspective diagram of lincomycin hydrochloride molecular of cryst

40、al form .44圖5鹽酸林可霉素晶型晶胞沿a軸的投影圖Figure 5: perspective diagram of crystal cell of lincomycin hydrochloride crystal form I along the a-axis.45 晶型II屬單斜晶系，空間群為P21 晶胞參數a=5.243(1)，b=33.894(2) ，c=13.633(1) ，=95.212(4)。每個晶胞內有4個鹽酸林可霉素分子，每個鹽酸林可霉素分子結合1個水分子與另一個一水鹽酸林可霉素分子復合，再結合0.5個水分子，即其化學計量式為(C18H34N2O6SHClH2O)2（

41、H2O）0.5；計算分子量為461.01。46 The crystal form II belongs monoclinic system, in space group P21, Z=4, with unit cell parameters: a=5.243(1)，b=33.894(2)，c=13.633(1);=95.212(4); stoichiometric formula: (C18H34N2O6SHClH2O)2 (H2O）0.5；，calculated molecular weight: 461.01. 其分子結構中五元環（古液酸）、六元環（林可霉糖）及1、4位取代基、2、3位及

42、5位取代基鍵構型雖均與晶型相同，但因不對稱單位中兩林可霉素的五元環空間取向不同，使其互為構象異構體而不能疊合。47 該晶型鹽酸林可霉素分子中O（4）在晶中占有兩個位置。C（4）,C(20),C(18),C(19)，C(20)原子因其部分位置無序，溫度因子稍高。該晶型無分子內氫鍵聯系；分子間氫鍵聯系為：N(10)O(5):(x-1,y,z)2.8730；O（5）Ow(2)（x,y,z）：2.6835；N(10)O(5)(x+1,y,z):2.9572;O(2)O(3):(x,y-1,z)2.8274;該晶型晶態下靠氫鍵力和范德華力維持空間的穩定排列。見下圖。 48圖6鹽酸林可霉素晶型不對稱單位中

43、兩個分子的疊合圖Figure 6: diagram of two molecules ply in the asymmetric unit of lincomycin hydrochloride crystal form 49圖7鹽酸林可霉素晶型的分子立體結構投影圖Figure 7: stereo-structure perspective diagram of lincomycin hydrochloride molecular of crystal form50圖8鹽酸林可霉素晶型晶胞內分子沿a軸方向的投影圖Figure 8: perspective diagram of crystal

44、cell of lincomycin hydrochloride crystal form along the a-axis.51 粉末射線衍射測定結果 Determination result by powder ray diffraction 經對晶型與晶型單晶X射線衍射計算，所得粉末X射線理論值及將其分別研碎后粉末X射線衍射實測值比較，晶型與晶型粉末X射線衍射譜的主要區別為：晶型在2角6.44、14.32和19.32處有強衍射峰，而晶型II則在2角5.18和10.4有強衍射峰。見下圖：52b. 晶型b. Crystal form 圖9 鹽酸林可霉素兩種晶型粉末的X射線衍射圖Figure

45、9： X-ray power diffraction pattern of polymorphs of lincomycin hydrochloride a. 晶型Ia. Crystal form I53圖10 鹽酸林可霉素原料的粉末射線衍射圖Figure 10: diagram of lincomycin hydrochloride drug substances by PXRD. 用來制備晶型、晶型單體的原料經X射線衍射分析，則系以晶型為主，同時含有少量晶型的混合物，其粉末X射線衍射圖如下：54 由圖可見， a. 晶型的主要衍射峰在原料粉末X射線圖中均明顯出現： Principal dif

46、fraction peak of crystal form I appear in X-ray power diffraction pattern of drug substances.55 b. 晶型的主要衍射峰在原料粉末X射線衍射圖中亦出現： Principal diffraction peak of crystal form II appear in X-ray power diffraction pattern of drug substances. 56紅外與近紅外光譜測定結果：（見下圖）。Fig11 infrared spectrum of crystal form I of li

47、ncomycin hydrochloride57 Fig12 infrared spectrum of crystal form II of lincomycin hydrochloride 58Fig13 near infrared spectrum of Crystal form I and form II of lincomycin hydrochloride59 鑒于晶型II比晶型I的實測效價總是高出大約50單位，兩種晶型均為有效晶型而且差別不大，根據我國20年來對其進行考察的實際結果，故將紅外分光光度法鑒別的壓片法改為石臘糊法，不再僅限定晶型I。 Because crystal fo

48、rm II potency more than crystal form I 50 units/mg, disparity between crystal form I and crystal form II nearly, thus determine that oil-mull technique replace pressed halide disk technique.60 HPLC法 High performance liquid chromatography 鹽酸林可霉素原料藥中可能會有7-差向林可霉素、a-酰胺差向林可霉素，林可霉素B、N-去甲基林可霉素等同系物，其抗菌活性均遠低

49、于林可霉素，我們經過四種HPLC法的試驗比較和色譜條件的優化，建立了符合ICH對分析方法要求的HPLC法，該法可分離出林可霉素；7-差向林可霉素、a-酰胺差向林可霉素，林可霉素B及待進一步確定結構的同系物等有關物質等6個有關物質，（見下圖）。61 Lincomycin hydrochloride may be accompanied 7-epilincomycin, a-amideepimer lincomycinB and lincomycin other homologous compounds, their potency are so lower than Lincomycin, we

50、 compared 4 HPLC methods by tests and optimized chromatography, the method can separate lincomycin, 7-epilincomycin, a-amideepime， lincomycin B and a lincomycin homologous compound etc.62圖14：林可霉素有關物質的HPLC圖Fig14：lincomycin hydrochloride related substances by HPLC63 檢查：水分 Water 鹽酸林可霉素兩種晶型熱重分析結果： 晶型I單晶

51、X射線衍射測定所得化學計量式為C18H34N2O6SHClH2O，依該式計算理論含水量為3.91%，我們用費休氏水分測定3次，所得均值為4.15%。采用熱重分析法其失重率為3.482%，均基本符合其理論值，見下圖：64 Determinate stoichiomertric formula of crystal form I is C18H34N2O6SHClH2O，theory content of water is 3.91%，determinate value is 4.15% by Karl Fischer method , lose weight is 3.482% by TGA,

52、they are conform to theory content of water.65圖15 鹽酸林可霉素兩種晶型的熱重分析圖Fig15：TGA curve of lincomycin hydrochloride two crystal forms.66 由圖可見晶型II的熱重曲線與晶型I不同點是它先失去一個分子水，爾后才失去兩個分子共用的0.5個分子水。 Two curves are different, which TGA curves shows that form II previously lose one crystalline water, hereafter lose h

53、alf crystalline water. 67 費休氏水分測定考察結果： 經我們考察12個生產廠家76批產品的結果，均為晶型為主，并存在少量晶型的混晶，但水分均在6.0%以內。 Examine 76 batches Of Lincomycin hydrochloride samples, that produced by 12 manufactory, which results shows their principal crystal forms are Form I, same time with a little of form II, but water contents are

54、 not more than 6.0%.68 有關物質 Related substances 制定藥品標準有關物質檢查項時應關注的問題： 1、該藥品中都有哪些物質或成分？ 2、都是什么物質或成分？其分子結構如何？ 3、這些物質或成分之間有無相互作用？ 4、這些物質或成分及其可能的相互作用產物對人體有什么作用？ 69 5、使用哪種方法能夠準確檢測它們？ 6、如何確定其限度？ Problems should be concerned the test method of relevant materials，when the work out drug standards: 1、What mate

55、rials or components are contained in drugs? 2、What are these materials or components? What are these molecules structure?70 3、Whether or not they are interaction between these materials or components? 4、What function of these substances or components and possible interaction product for human body?

56、5、What method can be used to test them accurately? 6、How restrict to theirs quantity?71 華北制藥集團新藥研究開發中心牛長群等近期采用液相色譜/質譜聯用儀對該廠林可霉素原料中的林可霉素、差向林可霉素、林可霉素B、差向林可霉素B及一個同系物做了HPLC與MS驗證，其HPLC（DAD）、ESI離子流圖與MS圖譜分列如下：72 Niu Changqun (pharmaceutical development centre of NCPC) measure lincomycin hydrochlorade , tha

57、t made in NCPC tructures of lincomycin and its related substances including: lincomycin, epilincomycin, lincomycinB, epilincomycinB and a lincomycin homologous compound by HPLC(DAD) and LC/MS, (see chart as below). 73 我們認為：在制定藥品標準時，對于林可霉素有關物質，由于相同分子量的同分異構體較多，（例如與林可霉素分子量相同者就有7-差向林可霉素、a-氨基林可霉素；與林可霉素B分

58、子量相同者就有N-去甲基林可霉素等），所以，他們的分子結構還需進一步確證。74 The author think: because molecules with same molecule weight are so more, in the related substances of lincomycin, (for example lincomycin and 7-epilincomycin, lincomycin and a-amideepimer; lincomycin B and N-demethyl lincomycin etc.), when work out drug standard, therefore theirs molecules struct