CN111929115A - 一种加热卷烟烟气捕集装置和加热卷烟样品前处理方法 - Google Patents
一种加热卷烟烟气捕集装置和加热卷烟样品前处理方法 Download PDFInfo
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Abstract
本发明属于加热烟具技术领域,特别涉及一种加热卷烟烟气捕集装置和加热卷烟样品前处理方法。所述烟气捕集装置包括:吸烟机,其内部具有剑桥滤片捕集器;捕集瓶,其连接在所述吸烟机的剑桥滤片捕集器之后,所述捕集瓶内下部分装有捕集液,所述捕集瓶的上部分具有周向贴在所述捕集瓶内壁的滤纸,所述滤纸整体被所述捕集液润湿。本发明还公开了一种加热卷烟样品前处理方法。本发明首次设计了一种模拟人体口腔的烟气捕集瓶,在捕集瓶的上部分内壁贴有被捕集液润湿的滤纸,捕集瓶设置在36.7‑37.7摄氏度的水浴装置,这样可以更真实的模拟人口腔粗糙湿润表面和口腔的唾液内吸收烟气的过程。
Description
技术领域
本发明属于加热烟具技术领域,特别涉及一种加热卷烟烟气捕集装置和加热卷烟样品前处理方法。
背景技术
烟草监管立法的日趋严格以及吸烟与健康研究的深入,导致了烟草公司寻求风险更低、无环境烟气的新型烟草制品。加热卷烟,其特征在于加热烟草而非燃烧烟草,向消费者提供一定的烟草特征感受。加热卷烟制品避免了传统卷烟制品燃烧时产生的复杂混合物所带来的危害,现阶段已成为国外烟草公司从传统卷烟制品转向的新方向之一。
目前加热卷烟用于生物学效应测试的样品主要是通过全烟气直接暴露和捕集烟气总粒相物(TPM,以下记为粒相物质)后的间接暴露。两种方式各有优缺点,全烟气直接暴露法,能更全面的反映样品的潜在效应,但因为全烟气直接暴露对仪器要求高,不利于推广。捕集TPM后的间接暴露,虽然捕集效率达到99%以上,但是仍有部分气相物质(GVP)无法捕集到剑桥滤片上。
在实现本发明的过程中,发明人发现:目前虽然也有针对气相物质进行捕集的方法,其是在剑桥滤片捕集器之后连接一个盛有磷酸缓冲溶液的冰浴玻璃捕集器。但是,进行细胞毒性测试时,为了方便,剑桥滤片捕集的TPM和冰浴玻璃捕集器捕集的GVP是分开进行细胞毒性检测实验的。
发明人发现上述方式存在以下问题:
1、冰浴玻璃捕集器的捕集液一般采用磷酸缓冲溶液,当在细胞毒性检测中,将捕集液加入细胞培养液中时,捕集液中的化学离子会影响细胞的正常生长,干扰细胞毒性检测结果。
2、由于TPM和GVP是分开进行细胞毒性检测实验的,因此无法得到单支卷烟烟气中的TPM和GVP整体对细胞生长的影响和毒性检测结果。因此,无法客观全面的评价加热卷烟烟气潜在的效应。
为了解决上述问题,提出本发明。
发明内容
针对现有技术的不足,本发明提供一种加热卷烟烟气捕集装置和的加热卷烟样品前处理方法。剑桥滤片捕集结合捕集瓶捕集,同时获取粒相物质和气相物质用于后端的生物学效应测试,从而客观全面的评价加热卷烟烟气潜在的效应。
本发明第一方面提供一种用于生物学效应测试的加热卷烟烟气捕集装置,所述烟气捕集装置包括:
吸烟机,其内部具有剑桥滤片捕集器;
捕集瓶,其连接在所述吸烟机的剑桥滤片捕集器之后,所述捕集瓶内下部分装有捕集液,所述捕集瓶的上部分具有周向贴在所述捕集瓶内壁的滤纸,所述滤纸整体被所述捕集液润湿。
此处润湿的滤纸的目的是:模仿人口腔的粗糙湿润表面结构,使得模拟人口腔粗糙湿润表面和口腔的唾液内吸收烟气的过程更加贴近真实情况。
优选地,所述滤纸下端不接触所述捕集液,所述滤纸完全覆盖在所述捕集瓶主体内壁未接触到所述捕集液的部分。
优选地,所述捕集瓶设置在36.7-37.7摄氏度的水浴装置中。此处的目的是:模拟人口腔的温度环境。
之前使用捕集瓶捕集烟气气相物质的条件是:捕集瓶在冰浴装置中,目的是为了使得气相物质冷凝。
优选地,所述捕集液为细胞培养基。当然,所述细胞培养基为液体细胞培养基。
本发明第二方面提供一种用于生物学效应测试的加热卷烟样品前处理方法,所述样品前处理方法使用第一方面所述烟气捕集装置,所述样品前处理方法包括以下步骤:
A、使用第一方面所述的吸烟机抽吸加热卷烟,所述加热卷烟产生烟气样品,所述烟气样品依次经过所述剑桥滤片捕集器和所述捕集瓶,所述烟气样品中的粒相物质被所述剑桥滤片捕集器中的剑桥滤片捕集,所述烟气样品中的气相物质被所述捕集瓶内的捕集液和滤纸捕集;
B、将捕集到粒相物质的所述剑桥滤片用二甲基亚砜(DMSO)浸泡,提取,得到含有粒相物质的二甲基亚砜溶液,作为粒相物质储存液;
将捕集到气相物质的所述滤纸用所述捕集瓶内的捕集液浸泡,提取,得到含有气相物质的捕集液;
C、根据细胞毒性预实验,将步骤B所述的粒相物质储存液稀释到粒相物质工作液的浓度,以所述剑桥滤片捕集器和所述捕集瓶捕集过程的卷烟抽吸数量为衡量尺度,根据所述粒相物质工作液的浓度,将所述含有气相物质的捕集液按照一定比例,用所述捕集液稀释,得到稀释后的气相物质的捕集液;
D、将步骤C所述粒相物质工作液和所述稀释后的气相物质的捕集液两者混合,用于后端的生物学效应测试。
优选地,所述步骤A中,使用权利要求1所述的吸烟机抽吸加热卷烟,使用多个含所述剑桥滤片捕集器的抽吸通道同时抽吸多支卷烟,所述的吸烟机的至少一个抽吸通道的所述剑桥滤片捕集器之后连接所述捕集瓶,所述剑桥滤片捕集器捕集过程的卷烟数量和所述捕集瓶捕集过程的卷烟数量相同或者不同。
优选地,所述捕集液为后端的生物学效应测试所用的测试细胞对应的细胞培养基。
优选地,步骤C中,根据所述粒相物质工作液的浓度,计算所述含有气相物质的捕集液稀释比例的方法如下:
将所述剑桥滤片捕集器捕集过程,抽吸卷烟的总数量记为b支;
将所述剑桥滤片捕集器捕集过程,所述剑桥滤片捕集到的粒相物质的总质量记为aug;
所述粒相物质工作液的浓度记为dug粒相物质/ml二甲基亚砜;
将所述步骤B中,捕集液内,气相物质的浓度记为e支卷烟/ml捕集液;
本文中,捕集液中,GVP浓度的计算方式为:抽吸的卷烟支数/捕集液体积,因此单位为:支/ml。
本发明具有以下有益效果:
1、本发明首次设计了一种模拟人体口腔的烟气捕集瓶,在捕集瓶的上部分内壁贴有被捕集液润湿的滤纸,捕集瓶设置在36.7-37.7摄氏度的水浴装置,这样可以更真实的模拟人口腔粗糙湿润表面和口腔的唾液内吸收烟气的过程。而现有技术直接用冰浴捕集器捕集烟气气相物质,从未想过对其进行改进以更加接近人体口腔吸收烟气的过程。
2、本发明首次使用生物学效应测试用的细胞对应的细胞培养基作为捕集瓶内的捕集液,这种方法避免了一般磷酸缓冲液捕集液中的离子对细胞生长的影响,完全不会对后端的生物学效应测试结果产生干扰。
3、本发明首次将含有TPM和GVP的两种样品液体按照一定的比例混合,再运用于后端测试时,能够客观有效地反映TPM和GVP的综合生物学效应。
此外,本发明首次考虑到对GVP进行按照抽吸的卷烟数量进行稀释,首次提出了计算稀释比例的方法,以所述剑桥滤片捕集器和所述捕集瓶捕集过程的卷烟抽吸数量为衡量尺度,根据所述粒相物质工作液的浓度,将所述含有气相物质的捕集液按照一定比例,用所述捕集液稀释,得到稀释后的气相物质的捕集液,用于后端的生物学效应测试。本方法避免了捕集的时候无限富集气相物质,导致捕集液的pH值过高,pH值影响细胞的存活率,无法检测物质本身的危害性的缺陷。
4、本发明首次综合考察了加热卷烟的暴露途径和作用方式,建立了较为完整的加热卷烟烟气前处理方法,将样品进行了有效处理,使得本发明方法获得的样品在运用于后端的生物学效应测试时,能够客观有效地反映加热卷烟烟气生物学效应的真实状态。
附图说明
图1为实施例1样品气相物(GVP)捕集瓶。
图2为实施例1中不同抽吸模式下加热卷烟样品和参比卷烟样品的TPM、GVP、TPM+GVP的细胞毒性测试结果图。
图3为对比例1的4个样品气相物质(GVP)的细胞毒性测试结果。
附图标记列表:
1、进气管,11、出气头,2、进出气管盖,3、出气管,31、膨大段,4、滤纸。
具体实施方式
下面通过具体实施方式进一步说明本发明的内容。
实施例1
一种用于生物学效应测试的加热卷烟烟气捕集装置,所述烟气捕集装置包括:
吸烟机,其内部具有剑桥滤片捕集器;
捕集瓶,其连接在所述吸烟机的剑桥滤片捕集器之后,所述捕集瓶内下部分装有捕集液,所述捕集瓶的上部分具有周向贴在所述捕集瓶内壁的滤纸4,所述滤纸4整体被所述捕集液润湿。
所述滤纸4下端不接触到所述捕集液,所述滤纸4完全覆盖在所述捕集瓶主体内壁未接触到所述捕集液的部分。
所述捕集瓶设置在37摄氏度的水浴装置中。所述捕集液为液体细胞培养基。
所述捕集瓶的具体设计如图1所示,包含上下插接的上部进出气管盖2和下部的捕集瓶主体。所述滤纸4周向贴在所述捕集瓶主体内壁。所述滤纸4下端的捕集液未在图1中示意出。
所述上部进出气管盖2包括:
球状膨大部分,其为蘑菇状,具有蘑菇头和蘑菇杆,上端的蘑菇头密封,下端的蘑菇杆开放且与所述捕集瓶主体插接;
进气管1,所述进气管1穿过所述球状膨大部分,所述进气管1上端伸出所述上部进出气管盖2,用于与外界的进气通道相连,下端深入到所述捕集瓶主体的内部,所述进气管1下端部具有与管体一体连接的出气头11,所述出气头11为空心球状,球的表面具有若干个出气孔;
出气管3,所述出气管3与所述球状膨大部分的蘑菇头连接,并伸出所述上部进出气管盖2,所述出气管3上具有膨大段31。
所述出气管3上的膨大段31的一个作用是:安装时,便于与所述进气管1区分。
所述出气头11浸没在所述捕集液中,所述出气头11设计是为了使得喷出的气体更加分散,有利于所述捕集瓶内的捕集液更充分地吸收气体。
捕集瓶捕集烟气样品中GVP的过程如下:
使用时,外界的进气通道与所述进气管1上端连接,烟气样品从进气通道进入所述进气管1,经过所述出气头11球状表面的若干个出气孔喷出到所述捕集瓶内的捕集液中,部分烟气样品被所述捕集液吸收,其余的烟气样品扩散到所述捕集瓶上部的空腔部分,有一部分烟气样品再次被捕集瓶内壁润湿的滤纸4所吸收,剩余的烟气样品通过所述球状膨大部分和出气管3扩散到所述捕集瓶外。
实施例2
一种用于生物学效应测试的加热卷烟样品前处理方法,具体为以下步骤:
制备加热卷烟样品和参比卷烟的TPM、TPM+GVP样品。加热卷烟样品为1#、参比卷烟样品为2#。2种待测样品的制备方法及抽吸参数见表1。
表1.2种待测样品的制备方法及抽吸参数
具体样品前处理方法如下:
1、根据加热卷烟加热时间和抽吸模式确定抽吸口数。
在本实施例中待测的两个样品在不同抽吸模式下的抽吸口数见表2,加热烟的抽吸口数为设定值,参比卷烟的抽吸口数为实际抽吸值。
表2两个样品在不同抽吸模式下的抽吸口数
2、用吸烟机抽吸,同时收集每个样品的TPM与相应的GVP。其中GVP用捕集瓶捕集(捕集瓶如图1所示),捕集液为培养基,TPM用剑桥滤片捕集。
3、按照表3样品制备关键参数进行捕集。
表3卷烟样品制备关键参数
4、抽吸过程中记录抽吸口数。
将捕集到粒相物质的所述剑桥滤片用二甲基亚砜(DMSO)浸泡,提取,得到含有粒相物质的二甲基亚砜溶液,作为粒相物质储存液;
将捕集到气相物质的所述滤纸用所述捕集瓶内的捕集液浸泡,提取,得到含有气相物质的捕集液。
(1)样品1#-I:
剑桥滤片捕集器捕集过程:
抽吸18支加热卷烟,抽吸口数为5口/支;
使用剑桥滤片6片,剑桥滤片共捕集到的TPM质量为0.47g,萃取使用DMSO体积为23.5ml,萃取后DMSO中TPM浓度为20mg/ml,作为储存液。待使用时,根据细胞毒性预实验,根据卷烟样品TPM的毒性,将TPM浓度稀释到合适的剂量浓度为600ug/ml,作为工作液,进行细胞毒性测试。
捕集瓶捕集过程:
抽吸12支加热卷烟,抽吸口数为5口/支,捕集液体积为20ml,GVP浓度为0.6支/ml。
每个样品的抽吸测试过程中,使用剑桥滤片根据烟支的抽吸数量进行更换,捕集瓶仅用一个,不再更换。
(2)样品1#-H:
剑桥滤片捕集器捕集过程:
抽吸18支加热卷烟,抽吸口数为10口/支;
使用剑桥滤片6片,剑桥滤片共捕集到的TPM质量为0.5212g,萃取使用DMSO体积为26.1ml,萃取后DMSO中TPM浓度为20mg/ml,作为储存液。待使用时,根据细胞毒性预实验,根据卷烟样品TPM的毒性,将TPM浓度稀释到合适的剂量浓度为600ug/ml,作为工作液,进行细胞毒性测试。
捕集瓶捕集过程:
抽吸12支加热卷烟,抽吸口数为10口/支,捕集液体积为20ml,GVP浓度为0.6支/ml。
(3)样品2#-I:
剑桥滤片捕集器捕集过程:
抽吸30支加热卷烟,抽吸口数为8.5口/支;
使用剑桥滤片6片,剑桥滤片共捕集到的TPM质量为0.2485g,萃取使用DMSO体积为24.9ml,萃取后DMSO中TPM浓度为10mg/ml,作为储存液。待使用时,根据细胞毒性预实验,根据卷烟样品TPM的毒性,将TPM浓度稀释到合适的剂量浓度为200ug/ml,作为工作液,进行细胞毒性测试。
捕集瓶捕集过程:
抽吸5支加热卷烟,抽吸口数为8.5口/支,捕集液体积为50ml,GVP浓度为0.1支/ml。
(4)样品2#-H:
剑桥滤片捕集器捕集过程:
抽吸18支加热卷烟,抽吸口数为13.2口/支。
使用剑桥滤片6片,剑桥滤片共捕集到的TPM质量为0.4328g,萃取使用DMSO体积为43.3ml,萃取后DMSO中TPM浓度为10mg/ml;作为储存液。待使用时,根据细胞毒性预实验,根据卷烟样品TPM的毒性,将TPM浓度稀释到合适的剂量浓度为200ug/ml,作为工作液,进行细胞毒性测试。
捕集瓶捕集过程:
抽吸3支加热卷烟,抽吸口数为8.5口/支,捕集液体积为30ml,GVP浓度为0.1支/ml。
5、以所述剑桥滤片捕集器和所述捕集瓶捕集过程的卷烟抽吸数量为衡量尺度,根据所述粒相物质工作液的浓度,将4个所述含有气相物质的捕集液样品按照一定比例,用液体细胞培养基稀释,得到稀释后的气相物质的捕集液;
将所述粒相物质工作液和所述稀释后的气相物质的捕集液两者混合,得到TPM+GVP样品,用于后端的生物学效应测试。
其中,TPM是用DMSO萃取的,表征单位为:ug粒相物质/ml二甲基亚砜。而GVP是由液体细胞培养基捕集,表征单位为:支卷烟/ml捕集液。
GVP稀释倍数的计算方法如下:以样品1#-I为例
根据本领域的常规方法,经过细胞毒性预实验,样品1#-I的TPM样品工作液浓度为600ug/ml,检测浓度为0ug/ml,60ug/ml,120ug/ml,180ug/ml,240ug/ml,320ug/ml,360ug/ml,420ug/ml,480ug/ml,540ug/ml,600ug/ml。,即在此范围内,细胞的存活率与TMP的浓度关联性较好。每个样品的工作液浓度和样品TPM的毒性有关。
样品1#-I的TPM浓度为:0.47*1000*1000/18=26111.11ug/支;
按卷烟支数折算,TPM浓度为:600/26111.11=0.0229787支/ml;
因为:GVP样品浓度为0.6支/ml,
所以:GVP样品稀释倍数为:0.6/0.0229787=26.11
因此,将GVP样品稀释26倍后,与TPM样品按照1:1的比例混合,进行测试。
稀释倍数的另一种计算方式是直接带入本发明的公式:
根据以上计算方法,样品1#-H的GVP样品稀释倍数为29,样品2#-I的GVP样品稀释倍数为4.14,样品2#-H的GVP样品稀释倍数为12。
实施例3
将实施例2步骤4制备获得的TPM工作液样品和步骤5制备的TPM+GVP样品进行细胞毒性测试。
测试方法为:MTT细胞毒性法。受试细胞为RPMI2650人鼻腔上皮细胞。
图2加热卷烟样品和参比卷烟的TPM、TPM+GVP样品对细胞存活率的影响
图2中,步骤4制备获得的TPM工作液样品记为:T,步骤5制备的TPM+GVP样品记为:T+V。横坐标表示所有样品中TPM的浓度,单位为:ug粒相物质/ml二甲基亚砜。
将实施例2步骤4制备获得的样品1#的TPM工作液样品和步骤5制备的TPM+GVP样品分别再次稀释到0~600ug粒相物质/ml二甲基亚砜(具体检测浓度为:0ug/ml、60ug/ml、120ug/ml、180ug/ml、240ug/ml、320ug/ml、360ug/ml、420ug/ml、480ug/ml、540ug/ml、600ug/ml)进行测试。
将实施例2步骤4制备获得的样品2#的TPM工作液样品和步骤5制备的TPM+GVP样品分别再次稀释到0~200ug粒相物质/ml二甲基亚砜(检测浓度为:0ug/ml、20ug/ml、40ug/ml、60ug/ml、80ug/ml、100ug/ml、120ug/m、l,40ug/ml、160ug/ml、180ug/ml、200ug/ml。)进行测试。稀释剂均为实施例2液体细胞培养基,来源是市售的RPMI1640细胞培养基。
由图2可以看出:
1、随着样品测试剂量的增高,受试细胞的细胞存活率呈现下降趋势。
2、TPM样品与TPM+GVP混合样品对受试细胞的存活率的影响无显著差异。这说明GVP对受试细胞的存活率的影响不明显。
这与本领域技术人员的认知一致:正常人抽吸的烟气通过剑桥滤片拦截后的气相物质,对细胞的存活率的影响应该微乎其微。
因此,GVP的稀释倍数的计算和TMP存储液的稀释倍数的计算,不能简单的按比例稀释,需要通过实验摸索和计算确定合适的比例。GVP和TMP两者之间的混合,不能简单的按比例稀释,需要通过实验摸索和计算确定合适的比例。
对比例1
将实施例2步骤5得到的4个样品的气相物质按照随机比例进行稀释处理,直接用于细胞毒性测试。
测试方法为:MTT细胞毒性法。受试细胞为RPMI2650人鼻腔上皮细胞。
图3为对比例1的4个样品气相物质(GVP)的细胞毒性测试结果。横坐标:剂量,单位:V捕集液/V稀释液(液体细胞培养基)%。纵坐标:细胞存活率,单位:%。
图3的结果:细胞存活率和稀释倍数的之间无特定的规律。随着稀释比例倍数的下降,其中2个样品(样品2#-I和2#-H)的细胞存活率急速下降到0,其中一个样品(样品1#-H)的细胞存活率下降缓慢,另外一个样品(样品1#-I)的细胞存活率甚至超过100%。
正常人抽吸的烟气剑桥滤片拦截后的气相物质,对细胞的存活率的影响应该微乎其微。因此,细胞存活率应接近100%。
本对比例1导致如图3的结果,可能原因是由于捕集的时候无限富集气相物质,导致捕集液的pH值过高,是pH值影响了细胞的存活率,而非物质本身的危害性。由此可见,捕集有GVP和TPM的两种样品混合时,GVP合适的稀释比例对于客观反映产品真实的安全性是必要的。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以权利要求的保护范围为准。
Claims (8)
1.一种用于生物学效应测试的加热卷烟烟气捕集装置,其特征在于,所述烟气捕集装置包括:
吸烟机,其内部具有剑桥滤片捕集器;
捕集瓶,其连接在所述吸烟机的剑桥滤片捕集器之后,所述捕集瓶内下部分装有捕集液,所述捕集瓶的上部分具有周向贴在所述捕集瓶内壁的滤纸,所述滤纸整体被所述捕集液润湿。
2.根据权利要求1所述的烟气捕集装置,其特征在于,所述滤纸下端不接触所述捕集液,所述滤纸完全覆盖在所述捕集瓶主体内壁未接触到所述捕集液的部分。
3.根据权利要求1所述的烟气捕集装置,其特征在于,所述捕集瓶设置在36.7-37.7摄氏度的水浴装置中。
4.根据权利要求1所述的烟气捕集装置,其特征在于,所述捕集液为细胞培养基。
5.一种用于生物学效应测试的加热卷烟样品前处理方法,其特征在于,所述样品前处理方法使用权利要求1所述烟气捕集装置,所述样品前处理方法包括以下步骤:
A、使用权利要求1所述的吸烟机抽吸加热卷烟,所述加热卷烟产生烟气样品,所述烟气样品依次经过所述剑桥滤片捕集器和所述捕集瓶,所述烟气样品中的粒相物质被所述剑桥滤片捕集器中的剑桥滤片捕集,所述烟气样品中的气相物质被所述捕集瓶内的捕集液和滤纸捕集;
B、将捕集到粒相物质的所述剑桥滤片用二甲基亚砜浸泡,提取,得到含有粒相物质的二甲基亚砜溶液,作为粒相物质储存液;
将捕集到气相物质的所述滤纸用所述捕集瓶内的捕集液浸泡,提取,得到含有气相物质的捕集液;
C、根据细胞毒性预实验,将步骤B所述的粒相物质储存液稀释到粒相物质工作液的浓度,以所述剑桥滤片捕集器和所述捕集瓶捕集过程的卷烟抽吸数量为衡量尺度,根据所述粒相物质工作液的浓度,将所述含有气相物质的捕集液按照一定比例,用所述捕集液稀释,得到稀释后的气相物质的捕集液;
D、将步骤C所述粒相物质工作液和所述稀释后的气相物质的捕集液两者混合,用于后端的生物学效应测试。
6.根据权利要求5所述的样品前处理方法,其特征在于,所述步骤A中,使用权利要求1所述的吸烟机抽吸加热卷烟,使用多个含所述剑桥滤片捕集器的抽吸通道同时抽吸多支卷烟,所述的吸烟机的至少一个抽吸通道的所述剑桥滤片捕集器之后连接所述捕集瓶,所述剑桥滤片捕集器捕集过程的卷烟数量和所述捕集瓶捕集过程的卷烟数量相同或者不同。
7.根据权利要求5所述的样品前处理方法,其特征在于,所述捕集液为后端的生物学效应测试所用的测试细胞对应的细胞培养基。
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