Article(id=1239231274481676827, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1239231265254207730, articleNumber=null, orderNo=null, doi=10.16155/j.0254-1793.2023-0531, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1692201600000, receivedDateStr=2023-08-17, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1773385359639, onlineDateStr=2026-03-13, pubDate=1725033600000, pubDateStr=2024-08-31, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773385359639, onlineIssueDateStr=2026-03-13, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773385359639, creator=13701087609, updateTime=1773385359639, updator=13701087609, issue=Issue{id=1239231265254207730, tenantId=1146029695717560320, journalId=1205117023404326918, year='2024', volume='44', issue='8', pageStart='1285', pageEnd='1462', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=0, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773385357440, creator=13701087609, updateTime=1773385579800, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1239232197937393856, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1239231265254207730, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1239232197937393857, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1239231265254207730, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1437, endPage=1442, ext={EN=ArticleExt(id=1239231274884330027, articleId=1239231274481676827, tenantId=1146029695717560320, journalId=1205117023404326918, language=EN, title=Determination of migration of 7 elements in recombinant human coagulation factor Ⅷ-Fc fusion protein for injection by ICP-MS, columnId=1206272757852074373, journalTitle=Chinese Journal of Pharmaceutical Analysis, columnName=Safety Monitoring, runingTitle=null, highlight=null, articleAbstract=
Objective:

To establish an inductively coupled plasma mass spectrometry (ICP-MS) method for the determination of migration Co,As,Br,Cd,Sb,Ba and Pb in recombinant human coagulation factor Ⅷ-Fc fusion protein for injection,and to quantitatively analyze the compatibility of long-term stored samples.

Methods:

After adding 0.75 mL of water to the sample for 4 h,the purified water was filled to 5 mL,and the contents of 7 elements in the solution were determined by standard addition method. The working parameters of ICP-MS:radio frequency (RF) power of 1 550 W,sampling depth of 10.0 mm,carrier gas flow rate of 0.50 L·min-1,atomizer pump speed of 0.30 r·s-1,atomization chamber temperature of 2 ℃,dilution gas flow rate of 0.74 L·min-1,repeated sampling times of 3 times,analysis mode of full quantitative.

Results:

The linear relationship of each element was good in the concentration range of the detected mass,and the recoveries of the element added were 92.0%-101.8%. When applied to the determination of the samples at each accelerated placement point,the increased migration amounts were 43.2,68.7 and 66.8 ng·mL-1,respectively,except the element content of Br increased slightly. The contents of the remaining 6 elements were far below the prescribed limit.

Conclusion:

The method is reliable and can be used for the determination of migration of 7 elements in recombinant human coagulation factor Ⅷ-Fc fusion protein for injection.

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目的:

建立电感耦合等离子体质谱(ICP-MS)法测定注射用重组人凝血因子Ⅷ-Fc融合蛋白中钴(Co)、砷(As)、溴(Br)、镉(Cd)、锑(Sb)、钡(Ba)、铅(Pb)的迁移量,并对相容性长期放置的样品进行定量分析。

方法:

样品加纯化水0.75 mL,倒置复溶4 h后,用纯化水定容至5 mL,采用标准加入法测定待测溶液中7个元素的含量。ICP-MS工作参数:射频功率为1 550 W,采样深度为10.0 mm,载气(氩气)流量为0.50 L·min-1,雾化器泵速为0.30 r·s-1,雾化室温度为2 ℃,稀释气流量为0.74 L·min-1,重复取样次数3次,分析模式为全定量。

结果:

各元素在各自的检测质量浓度范围内线性关系良好,元素加标回收率在92.0%~101.8%,应用于各加速放置点样品的检测时,除Br元素含量略有增长(增长迁移量分别为43.2、68.7、66.8 ng·mL-1)外,其余6个元素含量均远小于规定限度。

结论:

经方法学验证,本方法结果可靠,可用于注射用重组人凝血因子Ⅷ-Fc融合蛋白样品中7个元素的迁移量测定。

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Tel:13641986090;E-mail:

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The results of linear relation,LOD and LOQ

, figureFileSmall=null, figureFileBig=null, tableContent=
元素
(element)
线性范围
(linear range)/(ng·mL-1
回归方程
(regression equation)
rLOD/(ng·mL-1LOQ/(ng·mL-1
Co1~100Y=8.609×10-2X+1.038×10-30.999 80.0020.006
As1~100Y=1.114×10-2 X+6.113×10-41.0000.0060.021
Br10~1 000Y=2.930×10-4 X+8.868×10-40.999 92.7759.250
Cd1~100Y=4.783×10-3 X+1.860×10-40.999 90.0030.009
Sb1~100Y=3.507×10-3 X+1.037×10-40.999 90.0080.027
Ba1~100Y=1.009×10-3 X+7.463×10-50.999 60.0260.088
Pb1~100Y=8.393×10-3 X+8.207×10-40.999 70.0040.013
), ArticleFig(id=1239231279049274095, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231274481676827, language=CN, label=表1, caption=

线性关系、检测限及定量限结果

, figureFileSmall=null, figureFileBig=null, tableContent=
元素
(element)
线性范围
(linear range)/(ng·mL-1
回归方程
(regression equation)
rLOD/(ng·mL-1LOQ/(ng·mL-1
Co1~100Y=8.609×10-2X+1.038×10-30.999 80.0020.006
As1~100Y=1.114×10-2 X+6.113×10-41.0000.0060.021
Br10~1 000Y=2.930×10-4 X+8.868×10-40.999 92.7759.250
Cd1~100Y=4.783×10-3 X+1.860×10-40.999 90.0030.009
Sb1~100Y=3.507×10-3 X+1.037×10-40.999 90.0080.027
Ba1~100Y=1.009×10-3 X+7.463×10-50.999 60.0260.088
Pb1~100Y=8.393×10-3 X+8.207×10-40.999 70.0040.013
), ArticleFig(id=1239231279149937405, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231274481676827, language=EN, label=Tab.2, caption=

Recoveries and RSDs

, figureFileSmall=null, figureFileBig=null, tableContent=
元素
(element)
平均回收率
(average recovery)/%
重复性
(repeatability)RSD/%
Co95.11.4
As95.22.8
Br92.24.8
Cd95.40.36
Sb101.60.62
Ba101.81.6
Pb92.00.40
), ArticleFig(id=1239231279254795010, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231274481676827, language=CN, label=表2, caption=

回收率与重复性(n=6)

, figureFileSmall=null, figureFileBig=null, tableContent=
元素
(element)
平均回收率
(average recovery)/%
重复性
(repeatability)RSD/%
Co95.11.4
As95.22.8
Br92.24.8
Cd95.40.36
Sb101.60.62
Ba101.81.6
Pb92.00.40
), ArticleFig(id=1239231279355458313, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231274481676827, language=EN, label=Tab.3, caption=

The test results of samples

, figureFileSmall=null, figureFileBig=null, tableContent=
批号
(batch No.)
不同放置条件
(different placement condition)
含量(content)/(ng·mL-1
CoAsBrCdSbBaPb
107201704Z01B0个月(month)NDND140.1NDNDNDND
加速3个月(accelerated for 3 months)NDND164.9NDNDNDND
加速6个月(accelerated for 6 months)NDND183.3NDNDNDND
107201704Z02B0个月(month)NDND121.8NDNDNDND
加速3个月(accelerated for 3 months)NDND152.4NDNDNDND
加速6个月(accelerated for 6 months)NDND190.5NDNDNDND
107201705Z01B0个月(month)NDND131.3NDNDNDND
加速3个月(accelerated for 3 months)NDND170.8NDNDNDND
加速6个月(accelerated for 6 months)NDND198.1NDNDNDND
), ArticleFig(id=1239231279447733009, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231274481676827, language=CN, label=表3, caption=

样品测试结果

, figureFileSmall=null, figureFileBig=null, tableContent=
批号
(batch No.)
不同放置条件
(different placement condition)
含量(content)/(ng·mL-1
CoAsBrCdSbBaPb
107201704Z01B0个月(month)NDND140.1NDNDNDND
加速3个月(accelerated for 3 months)NDND164.9NDNDNDND
加速6个月(accelerated for 6 months)NDND183.3NDNDNDND
107201704Z02B0个月(month)NDND121.8NDNDNDND
加速3个月(accelerated for 3 months)NDND152.4NDNDNDND
加速6个月(accelerated for 6 months)NDND190.5NDNDNDND
107201705Z01B0个月(month)NDND131.3NDNDNDND
加速3个月(accelerated for 3 months)NDND170.8NDNDNDND
加速6个月(accelerated for 6 months)NDND198.1NDNDNDND
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ICP-MS测定注射用重组人凝血因子Ⅷ-Fc融合蛋白中7个元素的迁移量
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药物分析杂志 | 安全监测 2024,44(8): 1437-1442
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药物分析杂志 | 安全监测 2024, 44(8): 1437-1442
ICP-MS测定注射用重组人凝血因子Ⅷ-Fc融合蛋白中7个元素的迁移量
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丁豪
作者信息
  • 上海市食品药品包装材料测试所,上海 201203
  • Tel:13641986090;E-mail:

Determination of migration of 7 elements in recombinant human coagulation factor Ⅷ-Fc fusion protein for injection by ICP-MS
Hao DING
Affiliations
  • Shanghai Food and Drug Packaging Material Control Center,Shanghai 201203,China
出版时间: 2024-08-31 doi: 10.16155/j.0254-1793.2023-0531
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目的:

建立电感耦合等离子体质谱(ICP-MS)法测定注射用重组人凝血因子Ⅷ-Fc融合蛋白中钴(Co)、砷(As)、溴(Br)、镉(Cd)、锑(Sb)、钡(Ba)、铅(Pb)的迁移量,并对相容性长期放置的样品进行定量分析。

方法:

样品加纯化水0.75 mL,倒置复溶4 h后,用纯化水定容至5 mL,采用标准加入法测定待测溶液中7个元素的含量。ICP-MS工作参数:射频功率为1 550 W,采样深度为10.0 mm,载气(氩气)流量为0.50 L·min-1,雾化器泵速为0.30 r·s-1,雾化室温度为2 ℃,稀释气流量为0.74 L·min-1,重复取样次数3次,分析模式为全定量。

结果:

各元素在各自的检测质量浓度范围内线性关系良好,元素加标回收率在92.0%~101.8%,应用于各加速放置点样品的检测时,除Br元素含量略有增长(增长迁移量分别为43.2、68.7、66.8 ng·mL-1)外,其余6个元素含量均远小于规定限度。

结论:

经方法学验证,本方法结果可靠,可用于注射用重组人凝血因子Ⅷ-Fc融合蛋白样品中7个元素的迁移量测定。

电感耦合等离子体质谱  /  注射用重组人凝血因子Ⅷ-Fc融合蛋白  /  元素杂质  /  相容性  /  迁移量
Objective:

To establish an inductively coupled plasma mass spectrometry (ICP-MS) method for the determination of migration Co,As,Br,Cd,Sb,Ba and Pb in recombinant human coagulation factor Ⅷ-Fc fusion protein for injection,and to quantitatively analyze the compatibility of long-term stored samples.

Methods:

After adding 0.75 mL of water to the sample for 4 h,the purified water was filled to 5 mL,and the contents of 7 elements in the solution were determined by standard addition method. The working parameters of ICP-MS:radio frequency (RF) power of 1 550 W,sampling depth of 10.0 mm,carrier gas flow rate of 0.50 L·min-1,atomizer pump speed of 0.30 r·s-1,atomization chamber temperature of 2 ℃,dilution gas flow rate of 0.74 L·min-1,repeated sampling times of 3 times,analysis mode of full quantitative.

Results:

The linear relationship of each element was good in the concentration range of the detected mass,and the recoveries of the element added were 92.0%-101.8%. When applied to the determination of the samples at each accelerated placement point,the increased migration amounts were 43.2,68.7 and 66.8 ng·mL-1,respectively,except the element content of Br increased slightly. The contents of the remaining 6 elements were far below the prescribed limit.

Conclusion:

The method is reliable and can be used for the determination of migration of 7 elements in recombinant human coagulation factor Ⅷ-Fc fusion protein for injection.

inductively coupled plasma mass spectrometry(ICP-MS)  /  recombinant human coagulation factor Ⅷ-Fc fusion protein for injection  /  elemental impurity  /  package compatibility  /  migrating migration
丁豪. ICP-MS测定注射用重组人凝血因子Ⅷ-Fc融合蛋白中7个元素的迁移量. 药物分析杂志, 2024 , 44 (8) : 1437 -1442 . DOI: 10.16155/j.0254-1793.2023-0531
Hao DING. Determination of migration of 7 elements in recombinant human coagulation factor Ⅷ-Fc fusion protein for injection by ICP-MS[J]. Chinese Journal of Pharmaceutical Analysis, 2024 , 44 (8) : 1437 -1442 . DOI: 10.16155/j.0254-1793.2023-0531
人凝血因子Ⅷ(human coagulation factor Ⅷ,FⅧ)是人体内源性凝血系统级联反应中的一个重要因子,由肝脏中的肝窦内皮细胞和血管内皮细胞合成,是一种相对分子质量较大,由多肽前体演变而成活性杂合双体的糖蛋白,在止血过程中发挥着至关重要的作用[1-3]。由于FⅧ缺乏引起的血友病A(haemophilia A),是临床上较为普遍的血友病类型,约占血友病患者总数的80%,其中,男性发病率约为1/5 000[4]。血友病A目前尚无法根治,FⅧ进行替代治疗是现在的主要治疗方法[5]。FⅧ是从健康人血浆中提取制备的一种血液制品,但是近年来,由于血浆来源紧张以及血源类产品有潜在传播病毒的风险,重组人凝血因子Ⅷ(recombinant coagulation factor Ⅷ,rFⅧ)注射剂产品开始被研究生产并用于临床治疗[6-8]。rFⅧ通过基因重组技术获得,可有效降低人或动物来源成分,从而降低免疫原性及病毒污染的可能,提高产品的安全性[9]。rFⅧ用于血友病A,主要是在纠正或预防出血,急诊或择期手术中,可以起到暂时代替缺失的凝血因子的作用。目前,rFⅧ注射剂通常为冻干粉针剂,主要采用硼硅管制注射剂瓶和卤化丁基橡胶塞进行包装。本文采用ICP-MS法测定注射用rFⅧ-Fc融合蛋白中可能从玻璃迁移的钴(Co)、砷(As)、镉(Cd)、锑(Sb)、钡(Ba)、铅(Pb)6个元素,以及可能从溴化丁基橡胶塞中迁移的溴(Br)元素的迁移量。该方法操作简便,准确可靠,专属性强,可对注射用重组人凝血因子Ⅷ-Fc融合蛋白中可能迁移的重金属元素进行监控,为注射用重组人凝血因子Ⅷ-Fc融合蛋白的风险监控及相容性研究提供技术参考。
Agilent 8900型等离子体质谱仪,安捷伦科技有限公司;Binder FD240恒温干燥箱和Binder KBF-720恒温恒湿箱,宾得公司;Milli-Q制水机,默克公司。
Co、As、Cd、Sb、Ba、Pb 6个元素的混合标准溶液(100 μg·mL-1,国家钢铁材料测试中心钢铁研究总院,批号21DC302);Br标准溶液(100 μg·mL-1,中国计量测试技术研究院,批号1904);硝酸(65%,Merck公司,批号Z235341122);纯化水(电阻率为18 MΩ·cm)。
3批厂家提供的注射用rFⅧ-Fc融合蛋白样品,批号分别为107201704Z01B、107201704Z02B、107201705Z01B,规格为250 IU。根据药品相容性指导原则,按照加速放置条件(15 ℃±2 ℃),放置加速3、6个月。
以氩气为载气,氦气为碰撞气,取调谐溶液对仪器条件进行优化,参数为:功率1 550 W,采样深度10.0 mm,载气流量0.50 L·min-1,雾化器泵速0.30 r·s-1,雾化室温度2 ℃,稀释气流量0.74 L·min-1,重复取样3次,分析模式为全定量,测定时选取的元素质量数为59Co、75As、81Br、111Cd、121Sb、137Ba、208Pb。其中,59Co以45Sc作为内标,75As、81Br以72Ge作为内标,114Cd以115In、121Sb、137Ba作为内标,208Pb以103Rh作为内标,各元素的质谱图见图1
取6个元素混合标准溶液(100 μg·mL-1)1.0 mL和Br元素的标准溶液(100 μg·mL-1)1.0 mL,置100 mL量瓶中,用纯化水稀释至刻度,得到质量溶液为1 μg·mL-1的混合标准溶液。取混合标准溶液0.1、0.5、1.0、2.0、2.5、5.0、10.0 mL,分别置100 mL量瓶中,用纯化水稀释至刻度,得到质量浓度分别为1、5、10、20、25、50、100 ng·mL-1的对照品溶液。
取样品1瓶,加纯化水0.75 mL复溶,常温避光倒置4 h后,转移至5 mL量瓶中,用纯化水定容至刻度,待测,同法制备2份。
取“2.2.1”项下系列浓度对照品溶液进行测定,以元素响应值Y作为纵坐标,元素质量浓度X作为横坐标,进行线性回归。结果见表1,各元素在线性范围内精密度与稳定性较好。
取纯化水平行测定10次。根据C=3Sb/k得到元素检测限,C=10Sb/k得到元素定量限,结果见表1
取“2.2.1”项下20 ng·mL-1的对照品溶液,连续测定6次,计算RSD。结果显示,Co、As、Br、Cd、Sb、Ba、Pb的精密度RSD分别为0.80%、0.080%、3.4%、0.53%、0.45%、0.89%和0.79%,精密度良好。
取批号为107201704Z01B的样品6瓶,分别精密加入1 μg·mL-1的混合标准溶液1.0 mL,用纯化水稀释定容至20 mL,即得20 ng·mL-1的加标样品,进行测定,计算平均回收率,结果见表2。因样品中部分元素含量低于检测限,故本试验的重复性用6次回收率测定值的RSD表示。
任意取回收率项下的1瓶加标样品,在测定完后放置24 h后再次测定,计算RSD,结果显示,Co、As、Br、Cd、Sb、Ba、Pb的RSD分别为2.5%、2.1%、2.1%、2.9%、2.9%、3.6%和2.4%,稳定性良好。
取各批次样品的供试品溶液进行测定,结果见表3。根据ICH-Q3D的有关要求,As、Cd、Pb为1类元素,Co为2A类元素,Sb、Ba为3类元素,如通过注射给药途径进入人体,As、Cd、Pb、Co、Sb、Ba的日允许暴露量(PDE)分别为不得超过15、2、5、5、90、700 μg[10-12]。注射用rFⅧ-Fc融合蛋白每日最大用量为16瓶,按照30% PDE计算日摄入量控制阈值,每瓶用纯化水稀释定容至5 mL后测定,故限度为As不得超过56.25 ng·mL-1,Cd不得超7.5 ng·mL-1,Pb不得超过18.75 ng·mL-1,Co不得超过18.75 ng·mL-1,Sb不得超过337.5 ng·mL-1,Ba不得超过2 625 ng·mL-1。测定结果表明,不同时间加速放置的样品均符合规定。
本文考察的7个元素中,6个元素为ICH-Q3D所限制的金属元素,且是硼硅玻璃的的主要杂质成分,硼硅玻璃的质量及稳定性一定程度上会影响到药品的质量和稳定性[13-15],危害人体健康,因此,我国市售的药用玻璃包装必须符合国家YBB标准的相关规定[16]。Br元素是溴化丁基橡胶塞的主要成分,考察Br元素的含量变化,可以看出胶塞对药品是否稳定安全。
加速放置的目的是为了考察样品的长期稳定性和相容性,从样品测试结果可以看出,随着放置时间的延长,除Br元素以外的其余元素均未检出,远小于30% PDE,说明玻璃中的金属元素未发生明显迁移,注射用rFⅧ-Fc融合蛋白与玻璃包装相容性良好。同时,3批样品的Br元素在加速放置6个月后含量略有增长,但相对都较小,增量分别为43.2、68.7、66.8 ng·mL-1,说明胶塞基本稳定。因此说明,一般的中硼硅玻璃瓶和溴化丁基橡胶塞足以满足注射用rFⅧ-Fc融合蛋白装药需求。
ICP-MS法可以同时测定多种元素,具有灵敏度高,干扰少,线性宽等优点,已被广泛应用于中药样品重金属的残留分析[17-19]。本文采用ICP-MS法测定注射用rFⅧ-Fc融合蛋白中7个元素,方法简便,可操作性强,且准确可靠,专属性高。从样品分析结果看,样品测试结果稳定,且回收率较好,为rFⅧ的相容性研究和产品质量控制提供了一定的经验。
使用ICP-MS法分析时多数元素经常会受到不同程度的同量异位素干扰。本试验中的待测元素Br、Cd、Sb、Ba、Pb都有多个同位素,可按照丰度大、干扰小的原则来选定待测元素质量数[20]。以Cd为例,Cd的同位素有106Cd、108Cd、110Cd、111Cd、112Cd、113Cd、114Cd和116Cd等8种,其中106Cd、108Cd和116Cd的丰度均小于10%,在ICP-MS设置采集质量数时一般不予考虑,112Cd和114Cd的丰度均大于20%,但都存在Sn干扰,110Cd、111Cd和113Cd丰度均大于10%且小于20%,但110Cd和113Cd也都存在Sn干扰,因此综合考虑选定111Cd。Br、Sb、Ba元素也是同样的原理,分别选择81Br、121Sb、137Ba;Pb元素主要有4种同位素,分别为204Pb、206Pb、207Pb、208Pb,其中208Pb丰度最大,但因为本试验基质为纯化水体系,在试验中发现,Pb在采集时若仅选定208Pb,未开启干扰方程,样品的加标回收率会偏低,参考相关文献和标准[21],后续试验中勾选干扰方程,即Mc(208)=M(206)×1+M(207)×1+M(208)×1,样品的加标回收率良好。
综合上述最优仪器条件,本研究使用ICP-MS方法对注射用rFⅧ-Fc融合蛋白样品中7个元素的迁移量进行检测分析,线性拟合度良好,方法操作简单,结果准确稳定,灵敏度高,满足样品测定的要求,可为注射用rFⅧ-Fc融合蛋白的风险监控及相容性研究提供技术参考。
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doi: 10.16155/j.0254-1793.2023-0531
  • 接收时间:2023-08-17
  • 首发时间:2026-03-13
  • 出版时间:2024-08-31
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  • 收稿日期:2023-08-17
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    上海市食品药品包装材料测试所,上海 201203
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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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