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1、动植物、微生物来源的化学药品质量控制中动植物、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河北省药品检验所杨梁河北省药品检验所杨梁河北省药品检验所杨梁Hebei provincial Institute for drug control Hebei provincial
2、Institute for drug control Yang LiangYang Liang摘摘摘摘 要要要要ABSTRACTABSTRACT 科学合理地应用生物测定法、理化测定法，制订能科学合理地应用生物测定法、理化测定法，制订能科学合理地应用生物测定法、理化测定法，制订能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够有效控制药品质量的药品标准，不断提高药品的有效性够有效控制药品质量的药品标准，不断提高药品的有效性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恒的目标。和安全性是药品标准工作永恒的目标。和安全性是
3、药品标准工作永恒的目标。和安全性是药品标准工作永恒的目标。Work out drug standard Work out drug standard，that is able to control that is able to control quality of drugs effectively,to improve safety and quality of drugs effectively,to improve safety and effectiveness of drug substances and drug product,is effectiveness of drug
4、substances and drug product,is eternal goal for drug standard work.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控制药品质量的实质就是要控制原料药和制剂中控制药品质量的实质就是要控制原料药和制剂中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的药物活性成分分子在其有效期内符合临床需要的药的药物活性成分分子在其有效期内符合临床需要的药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、
5、治疗物构效关系并保持其物理稳定性、化学稳定性、治疗物构效关系并保持其物理稳定性、化学稳定性、治疗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。学稳定性、微生物学稳定性和毒理学稳定性。学稳定性、微生物学稳定性和毒理学稳定性。学稳定性、微生物学稳定性和毒理学稳定性。Control molecular of activity pharmaceutical Control molecular of activity pharmaceutical ingredient in the drug substances and products,in ingredien
6、t in the drug substances and products,in essence of control qualityessence of control quality，conform to structure-conform to structure-activity relationship and maintain its physical stability,activity relationship and maintain its physical stability,chemical stability,therapeutic stability,microbi
7、ological chemical stability,therapeutic stability,microbiological stability and toxicology stability,before term of validity.stability and toxicology stability,before term of validity.因此，我们认为在方法学研究中应该注意因此，我们认为在方法学研究中应该注意以下以下3 3点：点：1 1、生物测定法是不可替代的方法。、生物测定法是不可替代的方法。2 2、理化测定法检测结果必须体现药物的、理化测定法检测结果必须体现药物
8、的真实活性或效价。真实活性或效价。3 3、科学合理地综合运用生物测定法和理、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。化测定法，才能有效控制药品质量。Therefore we must be attention to following 3 Therefore we must be attention to following 3 problemsproblems，when study and design for methodology:when study and design for methodology:1.Biological assay and test i
9、s the irreplaceable 1.Biological assay and test is the irreplaceable method.method.2.Determination results of physical and 2.Determination results of physical and chemical assay must be indicating true activity or chemical assay must be indicating true activity or potency of drug.potency of drug.3.O
10、nly scientifically and reasonably apply 3.Only scientifically and reasonably apply biological and physical and chemical test methods,biological and physical and chemical test methods,drug quality is controlled effectively.drug quality is controlled effectively.一、生物测定法是无可替代的药品检测方法。一、生物测定法是无可替代的药品检测方法
11、。Biological assay is the irreplaceable method of drug test.药物的分子结构及其构型、构象决定着药物药物的分子结构及其构型、构象决定着药物药物的分子结构及其构型、构象决定着药物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，（特别是药物制剂中的生物学特性和理化特性，（特别是药物制剂中的生物学特性和理化特性，（特别是药物制剂中的生物学特性和理化特性，（特别是药物制剂中药物分子的结构是否稳定？其构型、构象对于治药物分子的结构是否稳定？其构型、构象对于治药物分子的结构是否稳定？其构型、构象对于治药物分子的结构是否稳定？其构型、构象
12、对于治疗或预防疾病是否有利？其降解产物、可能的外疗或预防疾病是否有利？其降解产物、可能的外疗或预防疾病是否有利？其降解产物、可能的外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？），而检测与来物质会对人体产生何种危害？），而检测与来物质会对人体产生何种危害？），而检测与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。能发生的不
13、良反应）最直接的方法就是生物测定法。能发生的不良反应）最直接的方法就是生物测定法。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 The molecular structure of API in the drug product and its configuration and conformation decided its biological,its configuration and conformation decided its biological,physical a
14、nd chemical characteristics,physical and chemical characteristics,(especially must be(especially must be attention for that,the structure and its configuration attention for that,the structure and its configuration and conformation whether or not stabilize and conformation whether or not stabilize？w
15、hether or not advantageous for the resist diseases whether or not advantageous for the resist diseases or prevent diseasesor prevent diseases？its degradation substances its degradation substances and foreign material how hazard for human bodyand foreign material how hazard for human body？)Biological
16、 assay and test are most direct)Biological assay and test are most direct methods,which can test and evaluate biological methods,which can test and evaluate biological effect(including therapeutic action and effect(including therapeutic action and undesirable action).undesirable action).1.1.生物测定法是直接
17、体现真实治疗作用的方法生物测定法是直接体现真实治疗作用的方法生物测定法是直接体现真实治疗作用的方法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 Biological assay is the method of directly reflecting a real cure action of drug product.reflecting a real cure action of drug product.缩宫素源自猪牛羊垂体后叶的提取物。是缩宫素源自猪牛羊垂体后叶的提取物。是缩宫素源自猪牛羊垂体后叶的提取物。
18、是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临床用于引产、产前子宫收缩不良和产后及子临床用于引产、产前子宫收缩不良和产后及子临床用于引产、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宫肌瘤术后的止血药，其低剂量可增强子宫节宫肌瘤术后的止血药，其低剂量可增强子宫节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律性收缩、高剂量则可使肌层内血管受压而止律性收缩、高剂量则可使肌层内血管受压而止律性收缩、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。血。因而至今在临床广泛应用。血。因而至今在临床广泛应用
19、。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高度与缩宫素检测时测定的子宫肌肉收缩的高度与缩宫素检测时测定的子宫肌肉收缩的高度与缩宫素检测时测定的子宫肌肉收缩的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的效价单位数相关显著。所以其大鼠离体子宫测的效价单位数相关显著。所以其大鼠离体子宫测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。定法直接代表了临床疗效。定法直接代表了临床疗效。定法直接代表了临床疗效。Oxytocin is extracted from hypophysinum of Oxytocin is extracted from hypophy
20、sinum of pig,cattle and sheep or goat.pig,cattle and sheep or goat.Oxytocin is treating for induction of labor,Oxytocin is treating for induction of labor,improve uterus contracts and postpartum styptic improve uterus contracts and postpartum styptic or after resection of uterus myoma,its lower dose
21、 or after resection of uterus myoma,its lower dose may impel the uterus contract,and its higher dose may impel the uterus contract,and its higher dose may control postpartum uterine bleeding by may control postpartum uterine bleeding by pressing vessel in muscle and therefore oxytocin is pressing ve
22、ssel in muscle and therefore oxytocin is applying widely in hospitals.applying widely in hospitals.The potency of oxytocin is estimated by The potency of oxytocin is estimated by comparing the contraction effect produced on comparing the contraction effect produced on isolated rat uterus with that p
23、roduced by isolated rat uterus with that produced by Posterior Pituitary or Oxytocin Standard.It Posterior Pituitary or Oxytocin Standard.It reflects the effect of clinic.The contraction of reflects the effect of clinic.The contraction of uterus has the remarkable correlation with the uterus has the
24、 remarkable correlation with the potency of oxytocin.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.表表1 1：缩宫素使子宫收缩高度发生变化的数据表：缩宫素使子宫收缩高度发生变化的数据表Table 1Table 1：date comp
25、aring the contraction effect produced on isolated rat uterus date comparing the contraction effect produced on isolated rat uterus with that produced by Posterior Pituitary or Oxytocin Standard.with that produced by Posterior Pituitary or Oxytocin Standard.表表2 2：缩宫素效价测定的生物统计和可靠性测验结果：缩宫素效价测定的生物统计和可靠性
26、测验结果Table 2Table 2：biological statistical results of the contraction effect produced on isolated rat biological statistical results of the contraction effect produced on isolated rat uterus with that produced by Posterior Pituitary with Oxytocin standard potency.uterus with that produced by Posterio
27、r Pituitary with Oxytocin standard potency.2 2、生物测定法是直接代表药品安全性的有效方法、生物测定法是直接代表药品安全性的有效方法、生物测定法是直接代表药品安全性的有效方法、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 Biological assay is the effective method that directly indicated the drug safety.directly indicated the drug safety.洋地黄毒苷是一
28、个源自紫花洋地黄的强心苷，洋地黄毒苷是一个源自紫花洋地黄的强心苷，洋地黄毒苷是一个源自紫花洋地黄的强心苷，洋地黄毒苷是一个源自紫花洋地黄的强心苷，其有效量为其有效量为其有效量为其有效量为0.050.05至至至至0.1mg0.1mg，其极量为，其极量为，其极量为，其极量为1.2mg1.2mg，即有，即有，即有，即有效量和中毒量之间距离很小，用鸽法进行生物测效量和中毒量之间距离很小，用鸽法进行生物测效量和中毒量之间距离很小，用鸽法进行生物测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，从定，可以直接有效地测定其效价和中毒剂量，从定，可以直接有效地测定其效价和中毒
29、剂量，从定，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而有效控制其制剂含量，为临床治疗慢性心衰并而有效控制其制剂含量，为临床治疗慢性心衰并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。防止洋地黄中毒，提供安全保证。防止洋地黄中毒，提供安全保证。防止洋地黄中毒，提供安全保证。Digitoxin is a kind of cardiac glycoside come Digitoxin is a kind of cardiac glycoside come from Digitalis purpurea L.Its effective
30、 dose is from Digitalis purpurea L.Its effective dose is from 0.05 to 0.1mg.Its maximum dose is 1.2mg,from 0.05 to 0.1mg.Its maximum dose is 1.2mg,The distance is so small from effective dose to The distance is so small from effective dose to poisoning dose.The potency of digitalis may be poisoning
31、dose.The potency of digitalis may be determined by comparing the minimum lethal determined by comparing the minimum lethal dose it produced by pigeons with that produced by dose it produced by pigeons with that produced by Digitalis Standard.This method can be used to Digitalis Standard.This method
32、can be used to effectively control the contents of a preparation of effectively control the contents of a preparation of digitalis and then give a safety to be a treatment to digitalis and then give a safety to be a treatment to heart failure and avoid digitalis poison.heart failure and avoid digita
33、lis poison.3 3、对于硫酸庆大霉素等多组分抗生素，由、对于硫酸庆大霉素等多组分抗生素，由、对于硫酸庆大霉素等多组分抗生素，由、对于硫酸庆大霉素等多组分抗生素，由于其各个组分于其各个组分于其各个组分于其各个组分HPLCHPLC法测定法的响应值与其法测定法的响应值与其法测定法的响应值与其法测定法的响应值与其效价的量效关系尚不明确，所以在用理化测效价的量效关系尚不明确，所以在用理化测效价的量效关系尚不明确，所以在用理化测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，还必须用微生定法控制其组分比例的同时，还必须用微生定法控制其组分比例的同时，还必须用微生定法控制其组分比例
34、的同时，还必须用微生物测定法测定各组分的总效价，以控制其总物测定法测定各组分的总效价，以控制其总物测定法测定各组分的总效价，以控制其总物测定法测定各组分的总效价，以控制其总体活性。体活性。体活性。体活性。Because relationship of these peaks Because relationship of these peaks responding by HPLC of every constitute with its responding by HPLC of every constitute with its dose-effect relationship of po
35、tency is not dose-effect relationship of potency is not explicitness in order to control biological activity,explicitness in order to control biological activity,therefore must be determinate total potency by therefore must be determinate total potency by microbiological assay of antibiotics,microbi
36、ological assay of antibiotics,simultaneously determinate proportion of simultaneously determinate proportion of constitutes by physical-chemical assay,for constitutes by physical-chemical assay,for example gentamicin sulfate etc,of multi-example gentamicin sulfate etc,of multi-constitute antibiotics
37、.constitute antibiotics.表表表表3 3：硫酸庆大霉素各组分和效价测定结果表：硫酸庆大霉素各组分和效价测定结果表：硫酸庆大霉素各组分和效价测定结果表：硫酸庆大霉素各组分和效价测定结果表Table 3Table 3：assay results of ratio of gentamicin components and total potency.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测
38、物的生物活性和安全性 The test results of physical and chemical determination must be truly embodied the biological activity and safety of the sample 1.1.为了保护环境，保护动物，我们应当努力研究为了保护环境，保护动物，我们应当努力研究为了保护环境，保护动物，我们应当努力研究为了保护环境，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。取代生物测定法的理化分析法。取代生物测定法的理化分析法。取代生物测定法的理化分析法。In order to protect t
39、he environment and animals,In order to protect the environment and animals,we should study physical and chemical analysis we should study physical and chemical analysis methods hard to instead of biological assays.methods hard to instead of biological assays.2.2.采用的理化测定法必须用生物测定法验证与采用的理化测定法必须用生物测定法验证
40、与采用的理化测定法必须用生物测定法验证与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评价，即理化测定法检测目标物及其测定结果必评价，即理化测定法检测目标物及其测定结果必评价，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。须能体现其生物活性。须能体现其生物活性。须能体现其生物活性。Analytical target components and determinable Analytical target components and determinable result by the physical and chemical determina
41、tion result by the physical and chemical determination method must be able to verify and evaluated by method must be able to verify and evaluated by biological assays,namely the results of physical biological assays,namely the results of physical and chemical determination must be able to and chemic
42、al determination must be able to embody the biological activity.embody the biological activity.FB0601 FB0601是一个是一个是一个是一个-内酰胺类半合成抗生素，内酰胺类半合成抗生素，内酰胺类半合成抗生素，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。其申报标准中的含量测定方法为中和滴定法。其申报标准中的含量测定方法为中和滴定法。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菌我们以金黄色葡萄球菌我们以金黄色葡萄球菌我们以金黄色葡萄球菌CMCCCMCC（B)2600
43、3B)26003为检定菌，为检定菌，为检定菌，为检定菌，pH6.5IIpH6.5II号培养基和号培养基和号培养基和号培养基和pH6.0pH6.0缓冲液，缓冲液，缓冲液，缓冲液，用微生物检定法测定了其效价，结果表明：用微生物检定法测定了其效价，结果表明：用微生物检定法测定了其效价，结果表明：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不同生产厂家样品的中和滴定法测定结果与不同生产厂家样品的中和滴定法测定结果与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。测得效价之间差异显著。测得效价之间差异显著。测得效价之间差异显著。FB0601 is a half
44、-synthetic-lactam antibiotic,FB0601 is a half-synthetic-lactam antibiotic,using neutralization titration to determine the using neutralization titration to determine the content in its application registered standard.With content in its application registered standard.With Staphyl.aureus CMCC(B)2600
45、3 as test organismStaphyl.aureus CMCC(B)26003 as test organism，pH6.5 II as medium pH6.5 II as medium，pH6.0 sterile bufferpH6.0 sterile buffer，its its titer evaluation by microbiological assay of titer evaluation by microbiological assay of antibiotics.The results show that antibiotics.The results sh
46、ow that：significant significant difference between titer evaluation and titration difference between titer evaluation and titration coming from different manufacturers ing from different manufacturers samples.继而，我们采用继而，我们采用继而，我们采用继而，我们采用ODSODS柱，以磷酸盐缓冲液：柱，以磷酸盐缓冲液：柱，以磷酸盐缓冲液：柱，以磷酸盐缓冲液：乙腈（乙腈（乙腈（乙腈（7575：
47、2525）为流动相，流速每分钟）为流动相，流速每分钟）为流动相，流速每分钟）为流动相，流速每分钟1ml,1ml,紫外紫外紫外紫外检测波长检测波长检测波长检测波长225nm225nm的的的的HPLCHPLC法检测，结果发现在法检测，结果发现在法检测，结果发现在法检测，结果发现在8 8批不同工艺的产品中，既有单个主要色谱峰批不同工艺的产品中，既有单个主要色谱峰批不同工艺的产品中，既有单个主要色谱峰批不同工艺的产品中，既有单个主要色谱峰(A)(A)者，也有两个主要色谱峰者，也有两个主要色谱峰者，也有两个主要色谱峰者，也有两个主要色谱峰(A(A、B)B)者。凡仅有单个者。凡仅有单个者。凡仅有单个者。凡
48、仅有单个色谱峰色谱峰色谱峰色谱峰A A者，其效价均远高于有两个色谱峰者，其效价均远高于有两个色谱峰者，其效价均远高于有两个色谱峰者，其效价均远高于有两个色谱峰A A、B B者，（见下图）。者，（见下图）。者，（见下图）。者，（见下图）。Then,we adopt HPLC,ODS column,Then,we adopt HPLC,ODS column,phosphate buffer:acetonitrile(75:25)as mobile phosphate buffer:acetonitrile(75:25)as mobile phase,flow ratephase,flow rate
49、：1ml/min,and determination 1ml/min,and determination wavelength 225nm by UV detector.We found both wavelength 225nm by UV detector.We found both single principal peak(A)and two principal peaks single principal peak(A)and two principal peaks(A and B)in eight production technology.Only(A and B)in eigh
50、t production technology.Only single principal peak(A),its potency are far than single principal peak(A),its potency are far than two principal peaks of A and B(see chart as two principal peaks of A and B(see chart as below).below).图图图图2 2：FB0601FB0601的的的的HPLCHPLC图。上图为仅有单一组分峰，下图为有两个主组分峰。图。上图为仅有单一组分峰，