中二个软代谢物也较稳定
[15,20]
(测定量变化<710%)。因此,常温下应在48h 内完成尿样测定,或先于-40℃下存
放而后测定。2.8 吸烟者和被动吸烟者尿样中烟碱及其代谢物含量比较
采用LC -MS/MS 法测定了10名日抽吸20支相同品牌卷烟的吸烟者和5名工作环境中存在ETS 的被动吸烟者24h 尿液中烟碱及其9种代谢物的浓度。结果(表5)表明:①虽然抽吸相同品牌相同支数的卷烟,但各吸烟者尿中烟碱及其9种代谢物的含量却存在较大差异,可能是抽吸习惯不同或烟碱实际摄入量
和代谢方式的差异[1,16]
造成的;②被动吸烟者24h 尿液中都检测出烟碱、可替宁、反3’-羟基可替宁,部分检出可替宁糖苷、降烟碱、烟碱氮氧化物,且前3种物质的浓度稍高,但都远远低于吸烟者。因此,烟碱、可替宁、反3’-羟基可替宁有可能作为人尿液中被动吸烟的生物标记物。
表2 烟碱及其代谢物的标准曲线、线性范围、相关系数、检测限
待测物①
线性方程
线性范围
(μg/L )相关系数
(R 2)检测限②
(μg/L )烟碱Y =6.7E -03X +0.0142.0～8000.00.99921.0可替宁
Y =4.6E -03X +4E -032.0～8000.01.00000.5反3’-羟基可替宁Y =2.9E -03X +2E -032.0～8000.01.00001.3烟碱糖苷Y =7.6E -03X +4E -031.0～4000.00.99990.4可替宁糖苷
Y =3.5E -05X +5E -042.0～8000.01.00000.5反3’-羟基可替宁糖苷Y =8.3E -04X +8E -055.0～4000.00.99992.7降烟碱Y =1.5E -03X +5E -031.0～4000.00.99960.2降可替宁Y =2.0E -04X +6E -0510.0～4000.00.99864.2可替宁氮氧化物Y =5.9E -04X +4E -051.0～4000.00.99930.4烟碱氮氧化物
Y =6.5E -03X +8E -03
1.0～4000.0
0.9993
0.1
注:①烟碱及其糖苷以烟碱-d 3作内标,可替宁及其糖苷、降烟碱、降可替宁、可替宁氮氧化物、烟碱氮氧化物以可替宁-d 3作内标,反3’-羟基可替宁及其糖苷的内标为反3’-羟基可替宁-d 3;②以信噪比S/N =3对应浓度确定。
2
4? 1994-2009 China Academic Journal Electronic Publishing House. All rights reserved. 6fc4a7d876a20029bd642d4a
表3 不同浓度下烟碱及其代谢物的精密度和回收率(n =6)
化合物浓度
(μg/L )精密度
(%)回收率
(%)烟碱
10003.1981506.694108.278可替宁10002.81011504.7104104.2109反3’-羟基可替宁10003.81031503.7102106.7106烟碱糖苷5003.71051503.7109103.9116可替宁糖苷10002.1841503.49510
9.3
93
化合物
浓度
(μg/L )精密度
(%)回收率
(%)反3’-羟基可替宁糖苷
5002.81001503.7100104.597降烟碱
5002.71021503.892107.496降可替宁
5003.5971503.0100107.3105可替宁氮氧化物
5003.3951503.591104.395烟碱氮氧化物
5002.6991504.411010
3.3
116表4 尿样中烟碱及其代谢物的测定量随存放条件的变化①
(%)
时间
温度NN ic
②
N ic NCot OHCot NNO C NO Cot OHCot -G Cot -G
N ic -G 48h
-40℃
5.54.05.6
6.73.05.84.91.3-1.82.94℃
-7.02.3-2.3-0.7-2.2-3.2-1.1-7.4-4.92.4室温
-6.13.1-1.00.1-3.2-3.0-0.5-6.2-5.35.1一周
-40℃-2.92.8-1.23.5-2.6-2.12.2-2.9-3.3-0.54℃-11.3-4.8-5.8-7.2-5.1-9.3-6-8.4-7-3.4室温
-15.6
-9.5
-7.6
-8.5
-9.3
-11.5
-10.6
-12.4
-13.7
-14.1
①(不同存放条件下的测定值-取样时的测定值)/取样时的测定值;②NN ic,降烟碱;N ic,烟碱;NCot,降可替宁;OHCot,反
3’-羟基可替宁;NNO,烟碱氮氧化物;CNO,可替宁氮氧化物;Cot,可替宁;N ic -G,烟碱糖苷;OHCot -G,反3’-羟基可替宁糖
苷;Cot -G,可替宁糖苷;下同。
表5 部分吸烟者和被动吸烟者尿液中的烟碱及其9种代谢物的浓度①
(μg/L )
尿样②
N ic Cot OHCot N ic -G Cot -G OHCot -G NN ic Ncot NNO C NO 1#
238.0
②
300.1452.058.847.230.114.428.241.230.62#324.8910.23040.0380.01648.0664.045.6102.8552.0332.63#736.0701.51072.0488.0920.0304.441.180.8612.0190.64#544.01092.21036.0343.2632.0181.242.276.41076.0114.65#228.0114.9164.062.0101.670.416.223.159.642.36#464.0839.22348.037.415
.01020.036.475.6288.0229.27#836.01491.85840.0420.01944.01776.050.0102.4420.0500.08#1176.0595.01428.0254.8784.0338.848.653.6375.6159.59#480.01274.3880.0636.0904.0184.056.617.2972.0179.510#177.2131.3366.454.046.054.74.918.318.031.711#10.021.639.2nd
③
5.1nd 3.4nd 2.6nd 12#28.019.240.8nd 8.4nd 2.5nd 1.6nd 13#18.418.755.2nd 7.2nd 9.3nd 5.6nd 14#7.410.320.6nd nd nd 4.7nd nd nd 15#
21.2
11.1
34.3
nd
nd
nd
nd
nd
2.0
nd
①3次平行测定的平均值;②1#～10#为吸烟者尿样,11#～15#为被动吸烟者尿样;③nd 未检出。
3
4? 1994-2009 China Academic Journal Electronic Publishing House. All rights reserved. 6fc4a7d876a20029bd642d4a
3 结论
LC -MS/MS 法可以同时测定吸烟者和被动吸烟
者尿液中烟碱及其9种代谢物,前处理简单,重复性好,准确性高,适合批量尿样中烟碱及其9种代谢物的快速分析;烟碱、可替宁、反3’-羟基可替宁有可能作为人体尿液中被动吸烟的生物标记物。
参考文献
[1] TR I KER A R.B i omarkers derived fr om nicotine and its
metabolites:A revie w [J ].Beitr Tabakfor I nt,2006,22:1472175.
[2] SCHERER G .B i omonit oring of inhaled comp lex m ixtures 2
Ambient air,diesel exhaust and cigarette s moke [J ].Exp Toxicol Pathol,2005,57:752110.
[3] SCHERER G,E NG L J,URBAN M ,et al .Relati onshi p
bet w een machine 2derived s moke yields and bi omarkers in cigarette s mokers in Ger many [J ].Regul Toxicol Phar m,2007,47:1712183.
[4] LANG ONE J J,Gjika H B,VUNAKI S H VAN.Analytical
Deter m inati on of N icotine and Related Compounds and their M etabolites[M ].Am sterda m:Elsevier,1999.
[5] HE I N R I CH 2RAMM R,W EG NER R,G ARDE A H,et al.
Cotinine excreti on (t obacco s moke bi omarker )of s mokers and non 2s mokers comparis on of GC 2M S and R I A results[J ].I nt J Hyg Envir on Health,2002,205:4932499.
[6] FEYERABE ND C,RUSSE LL M A.A rap id gas 2liquid chr o 2
mat ographic method for the deter m inati on of cotinine and nicotine in bi ol ogical fluids[J ].J Phar m Phar macol,1990,42:4502452.
[7] ALLE NA J J i,LAW S ON G M ,ANDERS ON R,et al .A
ne w gas chr omat ography ?mass s pectr ometry method f or si m ultaneous deter m inati on of t otal and free trans 23’2hydr oxy 2cotinine and cotinine in the urine of subjects receiving trans 2der mal nicotine[J ].Clin Che m,1999,45:1852191.[8] KI M I,DAR W I N W D,HUESTI S M A.
Si m ultaneous
deter m inati on of nicotine,cotinine,norcotinine,and trans 23’2hydr oxycotinine in hu man oral fluid using s olid phase extracti on and gas chr omat ography 2mass s pectr ometry[J ].J Chr omat ogr B,2005,814:2332240.
[9] BRUCKERT E,JACOB N,LAMA I RE L ,et al .Relati on 2
shi p bet w een s moking status and seru m li p ids in a hyperli p i 2de m ic populati on and analysis of possible conf ounding fact ors [J ].Clin Che m,1992,38:169821705.
[10]GHOSHEH O A,BROWNE D,ROGERS T,et al .A
si m p le high perfor mance liquid chr omat ographic method f or the quantificati on of t otal cotinine,t otal 3’2hydr oxycotinine and caffeine in the p las ma of s mokers[J ].J Phar m B i omed Anal,2000,23:5432549.
[11]JUN Y ANG,Y AN HU,J IBAO C A I,et al .A ne w molecu 2
larly i m p rinted poly mer f or selective extracti on of cotinine fr om urine sa mp les by s olid 2phase extracti on [J ].Anal B i oanal Che m,2006,384:7612768.
[12]BE NT LEY M C,ABRAR M ,KE LKM ,et al .Validati on of
an assay for the deter m inati on of cotinine and 32hydr oxycoti 2nine in hu man saliva using aut omated s olid 2phase extracti on and liquid chr omat ography with tande m mass s pectr ometric detecti on[J ].J Chr omat ogr B,1999,723:1852194.[13]T UOM I T,JOHNSS ON T,RE I JULA K .Analysis of nico 2
tine,32hydr oxycotinine,cotinine,and caffeine in urine of passive s mokers by HP LC 2tande m mass s pectr ometry [J ].Clin Che m,1999,45:216422172.
[14]HE AVNER D L,R I CHARDS ON J D,MORG AN W T,et
al .Validati on and app licati on of a method f or the deter m i 2nati on of nicotine and five maj or metabolites in s mokers ’urine by s olid 2phase extracti on and liquid chr omat ography 2tande m mass s pectr ometry[J ].B i omed Chr omat ogr,2005,19:3122328.
[15]MOYER T P,CHARLS ON J R,E NGER R J,et al .Si m ul 2
taneous analysis of nicotine,
nicotine metabolites and
t obacco alkal oids in seru m or urine by tande m mass s pec 2tr ometry,with clinically relevant metabolic p r ofiles [J ].Clin Che m,2002,48:146021471.
[16]MEGER M ,MEGER 2K OSSI E N I,SCHULER 2M ETZ A,et
al .Si m ultaneous deter m inati on of nicotine and eight nicotine metabolites in urine of s mokers using liquid chr omat ography 2tande m mass s pectr ometry[J ].J Chr omat B,2002,778:2512261.
[17]XU XU,I B A M M ,W E I SE L C P .Si m ultaneous and sensi 2
tive measure ment of anabasine,
nicotine,
and nicotine
metabolites in hu man urine by liquid chr omat ography 2tande m mass s pectr ometry [J ].Clin Che m,2004,50:232322330.
[18]PE LLEGR I N I M ,MARCHE I E,ROSSI S,et al .L iquid
chr omat ography electr os p ray i onizati on tande m mass s pec 2tr ometry assay f or deter m inati on of nicotine and metabolites,caffeine and arecoline in breast m ilk [J ].Rap id Commun Mass Spectr om,2007,21:269322703.
[19]BYRD G D,UHR I N GM S,DE BETH I ZY J D,et al .D irect
deter m inati on of cotinine 2N 2glucur onide in urine using ther 2mos p ray liquid chr omat ography /mass s pectr ometry [J ].B i olM ass S pectr om,1994,23:1032106.
[20]KI M I,HUESTI SMA.A validated method f or the deter m i 2
nati on of nicotine,cotinine,trans 23’2hydr oxycotinine,and norcotinine in hu man p las ma using s olid 2phase extracti on and liquid chr omat ography 2at m os pheric p ressure che m ical i onizati on 2mass s pectr ometry[J ].J Mass S pectr om,2006,41:8152821.
4
4? 1994-2009 China Academic Journal Electronic Publishing House. All rights reserved. 6fc4a7d876a20029bd642d4a
