中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2012年
4期
333-338
,共6页
杨睿悦%周伟燕%张天娇%国汉邦%李红霞%王抒%董军%陈文祥
楊睿悅%週偉燕%張天嬌%國漢邦%李紅霞%王抒%董軍%陳文祥
양예열%주위연%장천교%국한방%리홍하%왕서%동군%진문상
吸烟%可替宁%串联质谱法%色谱法,液相
吸煙%可替寧%串聯質譜法%色譜法,液相
흡연%가체저%천련질보법%색보법,액상
Smoke%Cotinine%Isotope dilution mass spectrometry%Liquid chromatography tandem mass spectrometry
目的 建立一种同位素稀释液相色谱串联质谱(ID-LC/MS/MS)测定血清可替宁的方法,以评价不同吸烟状况健康人的吸烟暴露水平分布状况.方法 以[D3]-可替宁作内标,乙腈沉淀蛋白、离心后吸取上清液、氮气挥干、流动相重组,液相分离后进人串联质谱分析；然后,采用多离子反应监测模式,以标准品制作标准曲线,结合同位素内标法定量建立同位素稀释液相色谱串联质谱法.用以对0 68、48.42、94.34、250.95、287.04 μg/L 5个水平血清样本进行5次重复分析,每次每种血清重复分析3份,以考察方法的精密度；同时测定与评价血清样本添加不同浓度标准品的加样回收率和样本在常温、4℃、-80℃保存的稳定性,以及2010年10月至12月期间94名健康志愿者在不同吸烟状况下的血清可替宁分布情况.并用Mann-Whitney U检验分析60名非吸烟、14名戒烟与20名吸烟健康志愿者血清可替宁含量的差异.结果 用ID-LC/MS/MS检测血清可替宁在本试验条件下分离良好,无内源性物质的干扰,具有较好的特异性；血清可替宁、内标峰面积比与可替宁浓度的线性相关系数≥0.9993;测定5个水平血清样本的总变异系数(CV)分别为4.71％、1.40％、1.98％、1.10％和1.03％；批内CV分别为2.19％、0.78％、0.75％、0.65％和0.67％,ID-LC/MS/MS的检测限(LOD)和定量限(LOQ)分别为0.013和0.050 μg/L,具有较高的灵敏度；3次试验的加样回收率范围为99.22％ ～ 102.67％;样本在常温条件下放置2d、4 ℃放置7d以及-80℃冻存保存3个月测定结果的准确度为99.28％～ 100.87％,批间CV均＜5％.检测94名健康人血清可替宁水平呈偏态和尖态分布(偏度2.71,峰度6.65),其中,20名吸烟者的可替宁浓度为116.40 (63.17 ～241.12) μg/L,14名已成烟者为0.67 (0.15～0.95) μg/L,60名非吸烟者为0.22 (0.15 ～0.42) μg/L;已戒烟者(Z=-2.12,P＜0.05)和吸烟者(Z=-6.67,P＜0.001)血清可替宁浓度分别显著高于非吸烟者.结论 建立的ID-LC/MS/MS测定血清可替宁方法操作简便、特异、灵敏,可望在吸烟暴露水平评价及暴露与疾病发生危险的研究中提供有效技术平台.
目的 建立一種同位素稀釋液相色譜串聯質譜(ID-LC/MS/MS)測定血清可替寧的方法,以評價不同吸煙狀況健康人的吸煙暴露水平分佈狀況.方法 以[D3]-可替寧作內標,乙腈沉澱蛋白、離心後吸取上清液、氮氣揮榦、流動相重組,液相分離後進人串聯質譜分析；然後,採用多離子反應鑑測模式,以標準品製作標準麯線,結閤同位素內標法定量建立同位素稀釋液相色譜串聯質譜法.用以對0 68、48.42、94.34、250.95、287.04 μg/L 5箇水平血清樣本進行5次重複分析,每次每種血清重複分析3份,以攷察方法的精密度；同時測定與評價血清樣本添加不同濃度標準品的加樣迴收率和樣本在常溫、4℃、-80℃保存的穩定性,以及2010年10月至12月期間94名健康誌願者在不同吸煙狀況下的血清可替寧分佈情況.併用Mann-Whitney U檢驗分析60名非吸煙、14名戒煙與20名吸煙健康誌願者血清可替寧含量的差異.結果 用ID-LC/MS/MS檢測血清可替寧在本試驗條件下分離良好,無內源性物質的榦擾,具有較好的特異性；血清可替寧、內標峰麵積比與可替寧濃度的線性相關繫數≥0.9993;測定5箇水平血清樣本的總變異繫數(CV)分彆為4.71％、1.40％、1.98％、1.10％和1.03％；批內CV分彆為2.19％、0.78％、0.75％、0.65％和0.67％,ID-LC/MS/MS的檢測限(LOD)和定量限(LOQ)分彆為0.013和0.050 μg/L,具有較高的靈敏度；3次試驗的加樣迴收率範圍為99.22％ ～ 102.67％;樣本在常溫條件下放置2d、4 ℃放置7d以及-80℃凍存保存3箇月測定結果的準確度為99.28％～ 100.87％,批間CV均＜5％.檢測94名健康人血清可替寧水平呈偏態和尖態分佈(偏度2.71,峰度6.65),其中,20名吸煙者的可替寧濃度為116.40 (63.17 ～241.12) μg/L,14名已成煙者為0.67 (0.15～0.95) μg/L,60名非吸煙者為0.22 (0.15 ～0.42) μg/L;已戒煙者(Z=-2.12,P＜0.05)和吸煙者(Z=-6.67,P＜0.001)血清可替寧濃度分彆顯著高于非吸煙者.結論 建立的ID-LC/MS/MS測定血清可替寧方法操作簡便、特異、靈敏,可望在吸煙暴露水平評價及暴露與疾病髮生危險的研究中提供有效技術平檯.
목적 건립일충동위소희석액상색보천련질보(ID-LC/MS/MS)측정혈청가체저적방법,이평개불동흡연상황건강인적흡연폭로수평분포상황.방법 이[D3]-가체저작내표,을정침정단백、리심후흡취상청액、담기휘간、류동상중조,액상분리후진인천련질보분석；연후,채용다리자반응감측모식,이표준품제작표준곡선,결합동위소내표법정량건립동위소희석액상색보천련질보법.용이대0 68、48.42、94.34、250.95、287.04 μg/L 5개수평혈청양본진행5차중복분석,매차매충혈청중복분석3빈,이고찰방법적정밀도；동시측정여평개혈청양본첨가불동농도표준품적가양회수솔화양본재상온、4℃、-80℃보존적은정성,이급2010년10월지12월기간94명건강지원자재불동흡연상황하적혈청가체저분포정황.병용Mann-Whitney U검험분석60명비흡연、14명계연여20명흡연건강지원자혈청가체저함량적차이.결과 용ID-LC/MS/MS검측혈청가체저재본시험조건하분리량호,무내원성물질적간우,구유교호적특이성；혈청가체저、내표봉면적비여가체저농도적선성상관계수≥0.9993;측정5개수평혈청양본적총변이계수(CV)분별위4.71％、1.40％、1.98％、1.10％화1.03％；비내CV분별위2.19％、0.78％、0.75％、0.65％화0.67％,ID-LC/MS/MS적검측한(LOD)화정량한(LOQ)분별위0.013화0.050 μg/L,구유교고적령민도；3차시험적가양회수솔범위위99.22％ ～ 102.67％;양본재상온조건하방치2d、4 ℃방치7d이급-80℃동존보존3개월측정결과적준학도위99.28％～ 100.87％,비간CV균＜5％.검측94명건강인혈청가체저수평정편태화첨태분포(편도2.71,봉도6.65),기중,20명흡연자적가체저농도위116.40 (63.17 ～241.12) μg/L,14명이성연자위0.67 (0.15～0.95) μg/L,60명비흡연자위0.22 (0.15 ～0.42) μg/L;이계연자(Z=-2.12,P＜0.05)화흡연자(Z=-6.67,P＜0.001)혈청가체저농도분별현저고우비흡연자.결론 건립적ID-LC/MS/MS측정혈청가체저방법조작간편、특이、령민,가망재흡연폭로수평평개급폭로여질병발생위험적연구중제공유효기술평태.
Objective To establish a method for measuring serum cotinine by isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) and provide an assay that can be applied to theevaluation of the level of smoke exposure and to the risk analysis of smoking related diseases.Methods Blood samples were collected from 94 apparently healthy subjects from October to December in 2010 and centrifuged,and the sera were separated.Serum samples were mixed with [ D3 ] -cotinine ( as the internal standard) and treated with acetonitriles to precipitate protein.After centrifugation,the supernatants were transferred and evaporated under a stream of nitrogen until dryness and reconstituted with mobile phase.Then the residuals were analyzed by LC/MS/MS system with multiple reaction monitor model; the concentration of cotinine were quantified by the isotope internal standard method and the stand curve was employed with a series of calibration.To estimate the precision of the method,five frozen serum pools were repeatedly analyzed in five runs,and every pool was analyzed in triplicate.In addition,the recovery rates were analyzed with the serum sample added with different levels of standard.The stability of cotinine in serum preserved at room temperature,4 ℃ and - 80 ℃,respectively.Finally,the levels of cotinine of 94 healthy subjects were measured to evaluate the distribution of cotinine with different smoke statuses.Results Serum cotinine measured by ID-LC/MS/MS was separated well with few interferences.The correlation coefficients between the peak area ratios and cotinine concentrations were higher than 0.9993.The values of within-run coefficients of variation (CV) of five frozen serum pools (0.68,48.42,94.34,250.95 and 287.04 μg/L) were 2.19％,0.78％,0.75％,0.65％ and 0.67％,respectively.The values of total CV were 4.71％,1.40％,1.98％,1.10％ and 1.03％,respectively.The limit of detection (LOD) and limit of quantitation ( LOQ ) were 0.013 and 0.050 μg/L,respectively.The analytical recoveries ranged from 99.22％ to 102.67％.The samples could maintain stability within 2 d at room temperature,7 d at 4 ℃ and 3 months at -80 ℃ resulting the accuracy of measurements from 99.28％ to 100.87％ and the CV＜5％.The levels of cotinine of 94 healthy subjects were measured and shown skewed and leptokurtic distribution.The concentrations of twenty smokers,fourteen former smokers and sixty non-smokers were 116.40 (63.17 -241.12),0.67 (0.15 - 0.95 ) and 0.22 (0.15 - 0.42 ) μg/L,respectively.Furthermore,the level of cotinine of former smokers (Z =-2.12,P ＜0.05) and smokers (Z =-6.67,P ＜0.001) were statistically higher than non-smokers.Conclusions An ID-LC/MS/MS method for serum cotinine detection has been established.It is hoped that the method will be applied to the assessment of smoke exposure and its association with the risks of smoking related diseases since it is simple,specific,precise,sensitive and accurate.
