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1、动植物、微生物来源的化学药品质量控制中生物测定法与理化测定法的合理应用Reasonably apply biological and physical-chemical assay and test for the drugs,which extracted from animal and plant and microbes河北省药品检验所杨梁Hebei provincial Institute for drug control Yang Liang,摘 要ABSTRACT 科学合理地应用生物测定法、理化测定法，制订能够有效控制药品质量的药品标准，不断提高药品的有效性和安全性是药品标准工作永
2、恒的目标。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.,控制药品质量的实质就是要控制原料药和制剂中的药物活性成分分子在其有效期内符合临床需要的药物构效关系并保持其物理稳定性、化学稳定性、治疗学稳定性、微生物学稳定性和毒理学稳定性。Control molecular of
3、 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 of validity.,因此
4、，我们认为在方法学研究中应该注意以下3点：1、生物测定法是不可替代的方法。2、理化测定法检测结果必须体现药物的真实活性或效价。3、科学合理地综合运用生物测定法和理化测定法，才能有效控制药品质量。,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 physical and chemical assa
5、y 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.,一、生物测定法是无可替代的药品检测方法。Biological assay is the irreplaceable method of drug test.,药物的分子结构及其构型、构象决定着药物的生物学特性和理化特性，（特别
6、是药物制剂中药物分子的结构是否稳定？其构型、构象对于治疗或预防疾病是否有利？其降解产物、可能的外来物质会对人体产生何种危害？），而检测与,评价其特性所导致的生物效应（包括对疾病的治疗作用与可能发生的不良反应）最直接的方法就是生物测定法。The molecular structure of API in the drug product and its configuration and conformation decided its biological,physical and chemical characteristics,(especially must be attention f
7、or that,the structure and its configuration,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 most direct methods,which can test an
8、d evaluate biological effect(including therapeutic action and undesirable action).,1.生物测定法是直接体现真实治疗作用的方法 Biological assay is the method of directly reflecting a real cure action of drug product.缩宫素源自猪牛羊垂体后叶的提取物。是临床用于引产、产前子宫收缩不良和产后及子宫肌瘤术后的止血药，其低剂量可增强子宫节律性收缩、高剂量则可使肌层内血管受压而止血。因而至今在临床广泛应用。,检测时测定的子宫肌肉收缩的
9、高度与缩宫素的效价单位数相关显著。所以其大鼠离体子宫测定法直接代表了临床疗效。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 may impel the uterus contract,and its higher do
10、se may control postpartum uterine bleeding by pressing vessel in muscle and therefore oxytocin is applying widely in hospitals.,The potency of oxytocin is estimated by comparing the contraction effect produced on isolated rat uterus with that produced by Posterior Pituitary or Oxytocin Standard.It r
11、eflects the effect of clinic.The contraction of uterus has the remarkable correlation with the potency of oxytocin.,图1：缩宫素使子宫收缩高度发生变化的记录比较图Figure 1：comparing the contraction effect produced on isolated rat uterus with that produced by Posterior Pituitary or Oxytocin Standard.,表1：缩宫素使子宫收缩高度发生变化的数据表Ta
12、ble 1：date comparing the contraction effect produced on isolated rat uterus with that produced by Posterior Pituitary or Oxytocin Standard.,表2：缩宫素效价测定的生物统计和可靠性测验结果Table 2：biological statistical results of the contraction effect produced on isolated rat uterus with that produced by Posterior Pituitar
13、y with Oxytocin standard potency.,2、生物测定法是直接代表药品安全性的有效方法 Biological assay is the effective method that directly indicated the drug safety.洋地黄毒苷是一个源自紫花洋地黄的强心苷，其有效量为0.05至0.1mg，其极量为1.2mg，即有效量和中毒量之间距离很小，用鸽法进行生物测定，可以直接有效地测定其效价和中毒剂量，从而有效控制其制剂含量，为临床治疗慢性心衰并防止洋地黄中毒，提供安全保证。,Digitoxin is a kind of cardiac glyc
14、oside 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 lethal dose it produced by pigeons with that produced by Digitali
15、s 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.,3、对于硫酸庆大霉素等多组分抗生素，由于其各个组分HPLC法测定法的响应值与其效价的量效关系尚不明确，所以在用理化测定法控制其组分比例的同时，还必须用微生物测定法测定各组分的总效价，以控制其总体活性。,Because rela
16、tionship 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 microbiological assay of antibiotics,simultaneously determinate proportion of constit
17、utes by physical-chemical assay,for example gentamicin sulfate etc,of multi-constitute antibiotics.,表3：硫酸庆大霉素各组分和效价测定结果表Table 3：assay results of ratio of gentamicin components and total potency.,二、理化测定法的检测结果必须真实体现被测物的生物活性和安全性 The test results of physical and chemical determination must be truly embo
18、died the biological activity and safety of the sample,1.为了保护环境，保护动物，我们应当努力研究取代生物测定法的理化分析法。In order to protect the environment and animals,we should study physical and chemical analysis methods hard to instead of biological assays.,2.采用的理化测定法必须用生物测定法验证与评价，即理化测定法检测目标物及其测定结果必须能体现其生物活性。Analytical target
19、 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 to embody the biological activity.,FB0601是一个-内酰胺类半合成抗生素，其申报标准中的含量测定方法为中和滴定法。我们以金黄色葡萄球
20、菌CMCC（B)26003为检定菌，pH6.5II号培养基和pH6.0缓冲液，用微生物检定法测定了其效价，结果表明：不同生产厂家样品的中和滴定法测定结果与测得效价之间差异显著。,FB0601 is a half-synthetic-lactam antibiotic,using neutralization titration to determine the content in its application registered standard.With Staphyl.aureus CMCC(B)26003 as test organism，pH6.5 II as medium，pH
21、6.0 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 samples.,继而，我们采用ODS柱，以磷酸盐缓冲液：乙腈（75：25）为流动相，流速每分钟1ml,紫外检测波长225nm的HPLC法检测，结果发现在8批不同工艺的产品中，既有单个主要色谱峰(A)
22、者，也有两个主要色谱峰(A、B)者。凡仅有单个色谱峰A者，其效价均远高于有两个色谱峰A、B者，（见下图）。,Then,we adopt HPLC,ODS column,phosphate buffer:acetonitrile(75:25)as mobile phase,flow rate：1ml/min,and determination wavelength 225nm by UV detector.We found both single principal peak(A)and two principal peaks(A and B)in eight production techno
23、logy.Only single principal peak(A),its potency are far than two principal peaks of A and B(see chart as below).,图2：FB0601的HPLC图。上图为仅有单一组分峰，下图为有两个主组分峰。Figure2：determined results of FB0601 by HPLC.top:single principal peak(A);below:two principal peaks(A and B).,显而易见，该HPLC法的专属性和与体外抗菌活性的相关性均高于中和法。Obviou
24、sly,the method of HPLC is better than titration method in specificity and correlativity of antimicrobial action in vitro.,三、科学合理地综合运用生物测定法和理化测定法Use reasonably and compositely the biological,physical,chemical assay and test,为了既保护环境、节约资源，又确保药品标准能对药品的有效性、安全性和质量稳定性（包括物理、化学、治疗学、微生物学和毒理学稳定性）实施有效控制，我们认为：在制
25、订或修订药品标准时，必须科学合理地综合运用生物测定法和理化测定法。,The author think:when we work out drug standard,we may use reasonably and compositely the biological testing methods and physical-chemical testing methods to make the validity,safety,and stability of drug quality for sure and to achieve the goal of protecting enviro
26、nment and resource economy at the same time.,1、不断改进理化测定法的专属性并采用生物测定法对其验证与评价。Improve continuously the specificity of physical-chemical method and use biological method to verify and evaluate（1）方法学研究的专属性不但是指可能存在的某组分（如杂质、降解物、基质等）时，对被分析物准确可靠测定的能力，还应指药物活性成分的分子必须是具有可靠临床疗效的特定构型、构象（包括多晶和异构）和足够的纯度，并具备经生物测定法实
27、验确证的控制能力。,The specificity of methodology research is not only means the ability to assess unequivocally the analysis in the presence of components which may be expected to be present,(typically these might include impurities,degraded materials,matrix,etc.),accurately dependably the analyzed substanc
28、e,but also the ability to control the molecule of active pharmaceutical ingredient to have special configuration and conformation that includes polymorph and isomer of molecule,and enough purity.At the same time the method should have definite proof by biological method.,（2）到目前为止，药物的构效关系都是用单晶X射线衍射法与
29、生物测定法结合而确定的。因此，结合临床治疗学效果、已有的构效关系和生物测定法对其进行验证与评价，可为分析方法学研究提供选择和改进的依据。,Current medicines structure-activity relationship is determined by single crystal X-ray diffraction and biological method,so combining clinic therapeutic effect with the existing structure-activity relationship and biological metho
30、d to verify and evaluate it may supply a choosing and improving foundation for analysis research.,（3）从对于药品的有效性、安全性和稳定性等都能够实施有效控制的目标来说，没有一种方法可以同时识别药物的生物及理化特性，换言之，没有一种方法是万能的。所以，只能综合应用生物测定法和理化测定法。,There is no all-powerful testing method to identify biological and physical-chemical characteristic of dru
31、g at the same time to the goal of controlling drug substances and drug products efficacy,safety,and stability.Wherefore we should do is to combine the biological testing method and physical-chemical testing method.,2、加强对生产过程中可能产生的有关物质包括原料和辅料的同系物、降解物、异构体等和外来物质（包括包装材料及提取、精制过程中混入的杂质等），尤其要格外关注HPLC法之紫外检测
32、器不能识别的有关物质的控制和预防。,We also need strengthen to reinforce the control possible relative materials include raw materials,homologous compound of accessories and degraded-materials,isomer and foreign material etc.,(that includes parking materials and impurity from extracting and refining processes),especi
33、ally pay attention to relative material that cant be identified by ultraviolet detector etc.of HPLC.,3、为了保护环境，节约资源和减少污染，应按真实、准确、安全、可靠与精简高效的方针，综合运用生物测定和理化测定法设计和制定药品标准。For protecting environment,resource economy and decrease polluter according to guideline principle,which conform to true,accurate,safe
34、ty,confidence,simplify and efficient,guiding principle,we may reasonably apply biological and physical and chemical assay and test,to design drug standards.,（1）鉴别 Identification IR法 Infrared spectroscopy 盐酸林可霉素具有多晶现象。两种晶型的红外光谱行为差异显著，为此，我们进行了晶体结构研究。Lincomycin hydrochloride is having monograph.Its pat
35、tern of two crystal forms are different on middle infrared spectrum,because we study crystal structure of its two crystal forms.,a晶型I.b.晶型a.crystal form b.crystal form图3盐酸林可霉素两种的扫描电镜照片Figure 3：photographs of Crystal polymorphs of lincomycin hydrochloride by scan electron microscopy.,单晶射线衍射测定结果 Resul
36、t by single crystal-ray diffraction 其晶型I属正交晶系的P22121空间群晶胞参数为a=6.1670(31)，b=18.5350(8)，c=20.6170(5)，每个晶胞内含有4个盐酸林可霉素分子与4个水分子，化学计量式为C18H34N2O6SHCLH2O，计算分子量为461.01。,林可霉素分子五元环（古液酸）为信封式构象，六元环（林可霉糖）为椅式构象，1位疏甲基，4位羟甲基为直立键，2、3位羟基及5位取代基均为平伏键。其分子内氢键为O（4）O(2);2.8295；分子间氢键为O（1）O（3）（1+x,y,z）:2.9235，水分子与林可霉素分子间氢键：O
37、（w1）O（2）(-2-x,-1/2-y,-1/2+z):2.8849。晶态下分子以氢键力和范德华力维持空间稳定排列，见下图。,The crystal form I belongs orthorhombic 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.,图4盐酸林可霉素晶型分子立体结构投影图Fi
38、gure 4：stereo-structure perspective diagram of lincomycin hydrochloride molecular of crystal form.,图5盐酸林可霉素晶型晶胞沿a轴的投影图Figure 5:perspective diagram of crystal cell of lincomycin hydrochloride crystal form I along the a-axis.,晶型II属单斜晶系，空间群为P21 晶胞参数a=5.243(1)，b=33.894(2)，c=13.633(1)，=95.212(4)。每个晶胞内有4个
39、盐酸林可霉素分子，每个盐酸林可霉素分子结合1个水分子与另一个一水盐酸林可霉素分子复合，再结合0.5个水分子，即其化学计量式为(C18H34N2O6SHClH2O)2（H2O）0.5；计算分子量为461.01。,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；，c
40、alculated molecular weight:461.01.其分子结构中五元环（古液酸）、六元环（林可霉糖）及1、4位取代基、2、3位及5位取代基键构型虽均与晶型相同，但因不对称单位中两林可霉素的五元环空间取向不同，使其互为构象异构体而不能叠合。,该晶型盐酸林可霉素分子中O（4）在晶中占有两个位置。C（4）,C(20),C(18),C(19)，C(20)原子因其部分位置无序，温度因子稍高。该晶型无分子内氢键联系；分子间氢键联系为：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
41、)O(3):(x,y-1,z)2.8274;该晶型晶态下靠氢键力和范德华力维持空间的稳定排列。见下图。,图6盐酸林可霉素晶型不对称单位中两个分子的叠合图Figure 6:diagram of two molecules ply in the asymmetric unit of lincomycin hydrochloride crystal form,图7盐酸林可霉素晶型的分子立体结构投影图Figure 7:stereo-structure perspective diagram of lincomycin hydrochloride molecular of crystal form,图8
42、盐酸林可霉素晶型晶胞内分子沿a轴方向的投影图Figure 8:perspective diagram of crystal cell of lincomycin hydrochloride crystal form along the a-axis.,粉末射线衍射测定结果 Determination result by powder ray diffraction 经对晶型与晶型单晶X射线衍射计算，所得粉末X射线理论值及将其分别研碎后粉末X射线衍射实测值比较，晶型与晶型粉末X射线衍射谱的主要区别为：晶型在2角6.44、14.32和19.32处有强衍射峰，而晶型II则在2角5.18和10.4有强
43、衍射峰。见下图：,b.晶型b.Crystal form 图9 盐酸林可霉素两种晶型粉末的X射线衍射图Figure 9：X-ray power diffraction pattern of polymorphs of lincomycin hydrochloride,a.晶型Ia.Crystal form I,图10 盐酸林可霉素原料的粉末射线衍射图Figure 10:diagram of lincomycin hydrochloride drug substances by PXRD.,用来制备晶型、晶型单体的原料经X射线衍射分析，则系以晶型为主，同时含有少量晶型的混合物，其粉末X射线衍射图如
44、下：,由图可见，a.晶型的主要衍射峰在原料粉末X射线图中均明显出现：Principal diffraction peak of crystal form I appear in X-ray power diffraction pattern of drug substances.,b.晶型的主要衍射峰在原料粉末X射线衍射图中亦出现：Principal diffraction peak of crystal form II appear in X-ray power diffraction pattern of drug substances.,红外与近红外光谱测定结果：（见下图）。,Fig11
45、 infrared spectrum of crystal form I of lincomycin hydrochloride,Fig12 infrared spectrum of crystal form II of lincomycin hydrochloride,Fig13 near infrared spectrum of Crystal form I and form II of lincomycin hydrochloride,鉴于晶型II比晶型I的实测效价总是高出大约50单位，两种晶型均为有效晶型而且差别不大，根据我国20年来对其进行考察的实际结果，故将红外分光光度法鉴别的压片
46、法改为石腊糊法，不再仅限定晶型I。Because crystal form 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.,HPLC法 High performance liquid chromatography 盐酸林可霉素原料药中可能会有7-差向林可霉素、a-酰胺差向林可
47、霉素，林可霉素B、N-去甲基林可霉素等同系物，其抗菌活性均远低于林可霉素，我们经过四种HPLC法的试验比较和色谱条件的优化，建立了符合ICH对分析方法要求的HPLC法，该法可分离出林可霉素；7-差向林可霉素、a-酰胺差向林可霉素，林可霉素B及待进一步确定结构的同系物等有关物质等6个有关物质，（见下图）。,Lincomycin hydrochloride may be accompanied 7-epilincomycin,a-amideepimer lincomycinB and lincomycin other homologous compounds,their potency are s
48、o lower than Lincomycin,we 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.,图14：林可霉素有关物质的HPLC图Fig14：lincomycin hydrochloride related substances by HPLC,检查：水分 Water 盐酸林可
49、霉素两种晶型热重分析结果：晶型I单晶X射线衍射测定所得化学计量式为C18H34N2O6SHClH2O，依该式计算理论含水量为3.91%，我们用费休氏水分测定3次，所得均值为4.15%。采用热重分析法其失重率为3.482%，均基本符合其理论值，见下图：,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.
50、482%by TGA,they are conform to theory content of water.,图15 盐酸林可霉素两种晶型的热重分析图Fig15：TGA curve of lincomycin hydrochloride two crystal forms.,由图可见晶型II的热重曲线与晶型I不同点是它先失去一个分子水，尔后才失去两个分子共用的0.5个分子水。Two curves are different,which TGA curves shows that form II previously lose one crystalline water,hereafter l
