Captiva EMR-Lipid技术结合超高效液相色谱-串联质谱测定血清中尼古丁和其代谢产物可替宁

目的 建立一种超高效液相色谱-串联质谱(UPLC-MS-MS)测定血清中尼古丁和可替宁的方法。方法 血清样品加入同位素内标后与乙腈混合均匀，经离心后得样品处理液，注入Captiva EMR-Lipid小柱，柱下连接0.22μm微孔滤膜，加压，净化，得最终样品。采用Poroshell 120 HILIC-Z色谱柱(2.1 mm×100 mm, 2.7μm),流动相为乙腈-10 mmol/L甲酸铵(pH 3.0)(V∶V为90∶10)等度洗脱，正离子扫描，内标法定量。结果 血清中可替宁和尼古丁在1.00～200 ng/mL范围内线性良好，检出限分别为0.01、0.066 ng/mL,定量限分别为0.033、0.2 ng/mL。可替宁的加标回收率为87.9%～114.2%,相对标准差(RSD)为3.9%～4.5%;尼古丁的加标回收率为86.4%～113.1%,RSD为4.2%～8.7%。结论 本方法简便、快速、高效，适用于血清中尼古丁和可替宁的测定。     

柱前衍生-超高效液相色谱-串联质谱法测定人体尿样中的对羟基苯甲酸

目的 建立丹磺酰氯柱前衍生-超高效液相色谱-串联质谱法测定人体尿液中对羟基苯甲酸的方法。方法 样品酶解后,丹磺酰氯衍生,乙腈提取。目标化合物采用Waters ACQUITY UPLC BEH C₁₈色谱柱(50 mm×2.1 mm, 1.7μm)分离,以乙腈和0.1%甲酸水溶液为流动相梯度洗脱,在电喷雾正离子源和多反应监测模式下进行测定,同位素内标法定量。结果 对羟基苯甲酸在5.0～1000μg/L浓度范围内线性关系良好,相关系数大于0.999,回收率为97.5%～102.0%,精密度为1.53%～5.80%(n=6),基质效应为100.7%～112.0%(n=6),检出限和定量限分别为0.5和1.5μg/L。应用本方法测定267份学生尿液样本,对羟基苯甲酸的检出率为100%,浓度范围为686～60842μg/L。结论 该方法有效提高定性准确性,可用于人体尿液中对羟基苯甲酸的定量分析。     

头发中尼古丁和可替宁的超高效液相色谱-串联质谱测定法

目的建立头发中尼古丁和可替宁的超高效液相色谱串联质谱(UPLC-MS-MS)测定方法。方法头发样品用Na OH溶液完全消解后用二氯甲烷+甲醇(3+1)萃取,以10 mmol/L乙酸铵(含0.1%甲酸)-乙腈作为流动相进行梯度洗脱,超高效液相色谱串联质谱多反应监测(MRM)模式测定,内标法定量。结果尼古丁在2~200μg/L的范围内,所得回归方程为y=1.296 7x-2.379 9,r=0.999 6;可替宁在0.2~20μg/L的范围内,所得回归方程为y=1.099 2x-0.005 15,r=0.999 9。以3倍信噪比计算,当取样量为50 mg时,尼古丁和可替宁检出限分别为0.01μg/g和0.001μg/g。该方法所得尼古丁的平均回收率在75.0%~97.0%之间,可替宁的平均回收率在90.0%~102.0%之间,RSD5%。结论本方法简便、快速、准确,适用于被动吸烟人群头发中尼古丁和可替宁含量的测定。     

尿液和头发中尼古丁和可替宁的毛细管气相色谱测定法

目的 建立尿液和头发中尼古丁和可替宁的气相色谱标准检测方法.方法 尿液或头发样品用NaOH溶液完全消解后用三氯甲烷萃取,经毛细管柱分离,NPD检测器检测,保留时问定性,以二苯胺作为内标物进行定量.结果 尿液中尼古丁和可替宁的检测范围分别为0.017～5μg/ml和0.024～5μg/ml,重复测定相对标准偏差(RSD...     

血清中的尼古丁和可替宁固相萃取-液相色谱-电喷雾离子阱串联质谱测定法

目的建立了血清中尼古丁和可替宁的固相萃取-液相色谱-电喷雾离子阱串联质谱测定方法,并测定血清中尼古丁和可替宁的含量。方法样品选用尼古丁和可替宁的d3同位素内标,经C18固相萃取小柱净化、富集后,经WatersSymmetry C18色谱柱分离,以甲醇0:.2%甲酸水溶液(体积比55:45)为流动相,流量为150μl/min。以氦气为碰撞气,正离子扫描方式测定血清中尼古丁和可替宁的含量。结果方法的线性范围为0.5～200.0 ng/ml,相关系数均为0.999 3,检出限均为0.50 ng/ml。样品加标回收率80.4%～103%,可替宁、尼古丁RSD分别为1.92%,0.64%(n=5)。结论本方法适用于血清中的尼古丁和可替宁的测定,具有分离效果好,稳定性强,操作简便、快捷,回收率高,重现性好等特点。     

超高效液相色谱-质谱法测定血浆和尿液中3-羟基异戊酰基肉碱

目的为建立生物素营养状况有效判断指标,利用超高效液相色谱-质谱联用仪开展血浆、尿样中3-羟基异戊酰基肉碱(3HIA-肉碱)含量测定方法的研究。方法取血浆、尿液样品3000×g高速冷冻离心10 min,采用MCX固相萃取柱进行分离纯化,经淋洗、洗脱、氮吹、复溶过程后测定。使用Waters Acquity UPLC BEH C_(18)(2.1 mm×50 mm,1.7μm)柱进行分离,以0.1%甲酸水溶液和甲醇为流动相进行梯度洗脱,在电喷雾正离子源(ESI~+)和多反应监测模式(MRM)下进行测定。采用同位素内标进行3HIA-肉碱的定性定量,并对所建立的方法进行精密度和准确度评价。结果血浆和尿液中3HIA-肉碱在0.1~20.0μg/L、5.0~200μg/L范围内呈良好的线性关系,R~20.99。3水平加标回收率78.6%~115.6%。血浆、尿液的RSD分别为4.80%、5.70%(n=6)。血浆中3HIA-肉碱检出限为0.04μg/L,定量限为0.10μg/L;尿液中3HIA-肉碱检出限为0.03μg/L,定量限为0.08μg/L。按该法测定人体尿液中3HIA-肉碱水平,其含量范围为55.50~313μg/L。结论本方法简便快捷,灵敏度和准确度较高,可满足研究生物体内血浆和尿液中3HIA-肉碱的需要。     

尿液中尼古丁和可替宁QuEChERS预处理气相色谱测定法建立

目的建立同时检测尿液中尼古丁和可替宁的QuEChERS样品预处理-气相色谱法。方法于2015年11月22日—12月12日,采集重庆医科大学6例吸烟者和10例不吸烟者尿液样本,通过QuEChERS提取和净化后,经非极性的DB-5 ms毛细管色谱柱分离,氢火焰离子化检测器(FID)检测,保留时间定性,峰面积定量。结果尿液中尼古丁和可替宁的线性范围分别为0.5~500μg/mL(r=0.999 8)和5~500μg/mL(r=0.998 9),最低检出限(LOD)分别为0.09和0.75μg/mL。2种待测物日内的相对误差(RE)和变异系数(CV)10.0%,日间RE和CV10.4%。尼古丁和可替宁的加标回收率分别在93.2%~107.8%和94.6%~99.2%范围内。结论建立的方法操作简单、快速、成本低且有机试剂用量少,适于暴露者的烟草暴露剂量评估。     

同位素内标稀释-液相色谱-串联质谱法同时测定猪组织中4种吩噻嗪类药物及其代谢物

目的 建立快速、准确、灵敏的猪组织(猪肉、猪肝和猪肾)中氯丙嗪、异丙嗪及其代谢产物氯丙嗪亚砜和异丙嗪亚砜的同位素内标稀释-液相色谱-串联质谱分析方法。方法 样品用乙腈提取,MCX固相萃取小柱净化。以0.1%甲酸乙腈和0.1%甲酸为流动相,采用梯度洗脱方式在ACQUITY UFLC?HSS T3色谱柱(100 mm×2.1 mm, 1.8μm)上进行分离,以电喷雾正离子多反应监测模式进行检测,同位素内标法定量。结果 氯丙嗪和异丙嗪在0.1～20.0μg/L,氯丙嗪亚砜和异丙嗪亚砜在0.5～100.0μg/L范围内具有良好的线性关系,相关系数(r)均大于0.999,方法的检出限为0.04～0.17μg/kg,定量限为0.12～0.51μg/kg,样品的回收率为90.8%～106.0%,精密度为1.9%～6.2%(n=6)。结论 本方法灵敏度高、结果准确,适用于猪肉、猪肝和猪肾中氯丙嗪、异丙嗪及其代谢产物氯丙嗪亚砜和异丙嗪亚砜残留的监测。     

同位素稀释-固相萃取-超高效液相色谱-串联质谱法测定食品中双酚A和双酚S

目的建立食品中双酚A及双酚S残留的同位素稀释-固相萃取-超高效液相色谱-串联质谱测定方法。方法样品加入同位素内标,经乙腈超声提取、ENVI Carb固相萃取柱净化后,以BEH C18(2.1 mm×100 mm,1.7μm)色谱柱分离,甲醇和水为流动相对目标物进行梯度洗脱。在电喷雾负离子多反应监测模式检测,内标法定量。结果双酚A和双酚S分别在0.5μg/L~50.0μg/L和0.1μg/L~10.0μg/L具有良好的线性关系,相关系数均0.999;双酚A在固态食品和液态食品中的定量限分别为0.6μg/kg和48 ng/L;双酚S在固态食品和液态食品中的定量限分别为0.1μg/kg和7.0 ng/L;低、中、高3个加标水平下,双酚A和双酚S在6类食品基质中的平均回收率为85.0%~108.4%,相对标准偏差(RSD)为0.7%~8.2%。结论该方法重现性好、灵敏度高、定量准确,可满足食品中双酚A和双酚S残留的检测需要。     

在线固相萃取-高效液相色谱-串联质谱法测定尿液中莠去津与呋喃丹

目的 建立尿液中莠去津与呋喃丹的在线固相萃取-高效液相色谱-串联质谱方法。方法 采用自制的纳米二氧化钛修饰共价有机框架材料作为在线固相萃取小柱的吸附填料,用于分离净化尿液中的痕量莠去津与呋喃丹;目标分析物在Shimpack XR-ODS II (150 mm×2.0 mm, 2.2μm)色谱柱上以含有0.1%甲酸的乙腈溶液和含有0.1%甲酸的水溶液为流动相进行分离,在电喷雾离子源（ESI）正离子多反应监测（MRM）模式下进行检测。结果 莠去津与呋喃丹在0.1μg/L～10.0μg/L具有良好的线性（r> 0.999 0）,检出限分别为0.015μg/L与0.012μg/L,定量限分别为0.05μg/L与0.04μg/L,加标回收率为86.2%～102%,相对标准差（RSD）为3.5%～6.3%。采用该方法对一例疑似莠去津中毒病人的尿液样品进行了测定,进一步验证了本方法在临床应用的可行性。结论 本法可用于尿液样品中残留痕量莠去津与呋喃丹的快速筛查与确证分析。     

Studies on a simultaneous analytical method of urinary nicotine and its metabolites, and their half-lives in urine

The objectives of this research were to improve and standardize a relatively easy, highly sensitive and highly accurate method of measuring nicotine, cotinine and trans-3'-hydroxycotinine in the urine of non-smokers exposed to environmental tobacco smoke (ETS) and to clarify the reliability of this method. Blinded studies using this analytical method were conducted in two universities. Standard solutions of nicotine, cotinine and trans-3'-hydroxycotinine were prepared at one university, divided in two parts and sent to another two universities for analyses by gas chromatography-mass spectrometry (GC/MS) without revealing the concentrations. It was found that the assay lower limit was at a level that could be used in passive smoking surveys and good results were obtained in crosschecks of samples of unknown concentration between the two universities. Since this method was considered to be useful for analyzing these urinary substances, ETS exposure experiments were performed in three universities using urinary nicotine, cotinine and trans-3'-hydroxycotinine as specific biomarkers of the urine. Non-smokers were exposed to ETS in an exposure room in each university. It was found that the nicotine concentrations in the urine of the subjects exposed to ETS reached a peak at about 2 hours after the end of exposure, which was somewhat later than that in active smokers. Because cotinine and trans-3'-hydroxycotinine in the urine are metabolites of nicotine, it was evident that the quantities were lower and the increasing rates were also less than that of nicotine. When the deceases in nicotine/ creatinine, cotinine/creatinine and trans-3'-hydroxycotinine/creatinine ratios in the urine were calculated using theoretical curves, the half-life times were calculated to be 13.9, 20.0 and 63.0 hours, respectively.     

Elimination of cotinine from body fluids: disposition in smokers and nonsmokers.

We have evaluated differences in the elimination of cotinine, a major nicotine metabolite, in smokers who quit smoking and never-smokers who were exposed to environmental tobacco smoke (ETS) under controlled conditions. The mean biological half-life of cotinine in urine, collected from the nine smokers was 16.5 +/- 1.2 h, in never-smokers exposed to ETS, 27.3 +/- 1.9 h. Differences in the mode of uptake and absorption of nicotine and possible differences in nicotine metabolism may play roles in the clearance rate differences between smokers and nonsmokers.     

Nicotine and cotinine in adults' urine: The German Environmental Survey 1998

In 1998, the German Environmental Survey (GerES III) recruited approximately 5000 adults between the ages of 18 and 69 years. The study population for these analyses consisted of 1580 smokers (34% of the total population) and 3126 nonsmokers. Nicotine and cotinine concentrations in urine were determined by HPLC methods with UV-detection and corrected for creatinine. Nicotine and cotinine concentrations differed between smokers and nonsmokers by factors of 10-100. The multiple linear regression models used for the analyses of nicotine detection in the urine of smokers explained 43.2% and 42.3% of the total volume-specific and creatinine-specific variances, respectively. Cigarette smoking was the major factor responsible for 41% of the total variance. The explained variances of the cotinine results were larger, 51.0% and 49.3% of the total variance were volume-specific and creatinine-specific, respectively. More than 20% of nonsmokers in GerES III were exposed to environmental tobacco smoke at home, at work or in other places. The logistic regression analysis approach used for the group of nonsmokers showed the greatest effects for those exposed to tobacco smoke at home (adjusted OR varied between 4 and 6). These results were seen for nicotine as well as for cotinine excretion. Exposure to tobacco smoke in the workplace doubled the risk for the detection of nicotine and cotinine in urine. When other risk factors such as age, sex, social status, community size, season of urine collection, and the consumption of food containing nicotine such as potatoes, cabbage, tea were included, the effect estimates for tobacco smoke exposure remained unchanged. A new federal bill to diminish environmental tobacco smoke (ETS) exposure in the workplace was recently passed in Germany, but protection of nonsmokers from smoking family members at home needs more attention.     

Passive and active maternal smoking as measured by serum cotinine: the effect on birthweight.

To determine how maternal exposure to environmental tobacco smoke affects birthweight, maternal sera obtained from 3529 pregnant women around 27 weeks gestation were analyzed for cotinine, a metabolite of nicotine. Based on cotinine levels, nonsmokers were divided into those exposed to environmental tobacco smoke (2-10 ng/mL) and those unexposed (< 2 ng/mL), and smokers were divided into tertiles. Compared with unexposed nonsmokers' infants, infants of exposed nonsmokers averaged 45 g less (P = .28) after adjustment for confounders, and smokers' infants averaged 78, 191, and 233 g less for the first, second, and third cotinine tertiles, respectively. Birthweight decreased 1 g for every nanogram per milliliter of cotinine increase (P < .001).     

Simultaneous measurement of urinary total nicotine and cotinine as biomarkers of active and passive smoking among Japanese individuals

Objectives

Measuring urinary cotinine is a popular and established method of biologically monitoring exposure to tobacco smoke. However, the lower detection limit of cotinine often impedes the evaluation of passive (second-hand) smoking and this, together with unconverted nicotine, does not reflect actual levels of exposure. Furthermore, a portion of the Japanese population might have decreased ability to metabolize nicotine. The present study was therefore carried out to validate the simultaneous analysis of total concentrations of free nicotine and cotinine and their glucuronides to determine actual levels of voluntary and involuntary exposure to cigarette smoke.

Methods

Urine samples from 118 Japanese smokers and 117 non-smokers were analyzed using gas chromatography–mass spectrometry. Voluntary and involuntary smoking status was self-reported and workplace smoking restrictions were objectively evaluated.

Results

The integrated sum of all concentrations showed 2.2- and 2.4-fold higher total levels (free and glucuronide) of nicotine and cotinine relative to the free levels. Median (quartiles) of total nicotine and cotinine were 1635 (2222) and 3948 (3512) ng/mL in smokers, and 3.5 (5.3) and 2.8 (4.2) ng/mL in non-smokers. Concentrations of urinary nicotine were higher than those of cotinine in 21 % of smokers and in 54 % of non-smokers. Nicotine and cotinine levels were significantly associated with a smoking habit, as well as being significantly associated with the workplace and home environments of non-smokers.

Conclusions

The present method can monitor voluntary and involuntary exposure to tobacco smoke. Measuring total urinary nicotine levels might be useful for analyzing exposure to cigarette smoke among non-smokers.     

Cord serum cotinine as a biomarker of fetal exposure to cigarette smoke at the end of pregnancy

This study investigated the association between biomarkers of fetal exposure to cigarette smoke at the end of pregnancy, cotinine in cord serum and in maternal and newborn urine samples, and quantitative measurement of smoking intake and exposure evaluated by maternal self-reported questionnaire. Study subjects were 429 mothers and their newborns from a hospital in Barcelona, Spain. A questionnaire including smoking habits was completed in the third trimester of pregnancy and on the day of delivery. Cotinine concentration in cord serum was associated with daily exposure to nicotine in nonsmokers and with daily nicotine intake in smokers. The geometric mean of cotinine concentration in cord serum statistically discriminated between newborns from nonexposed and exposed nonsmoking mothers, and between these two classes and smokers, and furthermore was able to differentiate levels of exposure to tobacco smoke and levels of intake stratified in tertiles. Urinary cotinine levels in newborns from nonsmoking mothers exposed to more than 4 mg nicotine daily were statistically different from levels in two other categories of exposure. Cotinine concentration in urine from newborns and from mothers did not differentiate between exposure and nonexposure to environmental tobacco smoke (ETS) in nonsmoking mothers. Cord serum cotinine appeared to be the most adequate biomarker of fetal exposure to smoking at the end of pregnancy, distinguishing not only active smoking from passive smoking, but also exposure to ETS from nonexposure.     

Secondhand Smoke Exposure and Urine Cotinine Concentrations by Occupation among Korean Workers: Results from the 2008 Korea National Survey for Environmental Pollutants in the Human Body

This study aimed to estimate the status of secondhand smoke (SHS) exposure through urine cotinine analysis among nonsmoking workers in Korea and to analyze factors affecting urine cotinine concentrations. Data were based on “The 2008 Korea National Survey for Environmental Pollutants in the Human Body,” a cross-sectional study of the National Institute of Environmental Research of Korea. We selected 1448 nonsmoking adult workers from 200 localities to participate in this survey. Urine cotinine concentrations were analyzed using a gas chromatograph-mass selective detector. We calculated separate covariate-adjusted geometric means for socio-demographic variables for males, females, and total subjects by analysis of covariance (ANCOVA). Statistical analyses were performed using SPSS version 18.0 (SPSS Inc., Chicago, Ill.). The prevalence of self-reported exposure to SHS was 36.9%. The geometric mean (95% confidence interval) of urine cotinine concentrations among all participants was 16.50 (14.48–18.80) μg/L. Gender, living area, education, and SHS exposure showed significant differences in urine cotinine concentrations. The urine cotinine concentrations of farmworkers and blue-collar workers such as skilled agricultural, forestry, and fishery workers, and elementary occupations were higher than those of white-collar workers such as clerical support workers, technicians, and associate professionals. Such a high proportion of the population having high urine cotinine levels indicates widespread exposure to SHS among nonsmoking workers in Korea. Furthermore, the urine cotinine levels among nonsmoking workers exposed to SHS varied by occupation. The measured urine cotinine concentration is suggested to be a valuable indication of SHS exposure in Korea.     

Is the hair nicotine level a more accurate biomarker of environmental tobacco smoke exposure than urine cotinine?

STUDY OBJECTIVE: The aim of this study was to compare the two biomarkers of exposure to environmental tobacco smoke (ETS); urine cotinine and hair nicotine, using questionnaires as the standard. DESIGN: A cross sectional study of children consecutively admitted to hospital for lower respiratory illnesses during the period of the study. SETTINGS: Three regional hospitals in the larger Wellington area, New Zealand. PARTICIPANTS: Children aged 3-27 months and admitted to the above hospitals during August 1997 to October 1998. A total of 322 children provided 297 hair samples and 158 urine samples. MAIN RESULTS: Hair nicotine levels were better able to discriminate the groups of children according to their household's smoking habits at home (no smokers, smoke only outside the home, smoke inside the house) than urine cotinine (Kruskall-Wallis; chi(2)=142.14, and chi(2)=49.5, respectively (p<0.0001)). Furthermore, hair nicotine levels were more strongly correlated with number of smokers in the house, and the number of cigarettes smoked by parents and other members of the child's households. Hair nicotine was better related to the questionnaire variables of smoking in a multivariate regression model (r(2)=0.55) than urine cotinine (r(2)=0.31). CONCLUSIONS: In this group of young children, hair nicotine was a more precise biomarker of exposure to ETS than urine cotinine levels, using questionnaire reports as the reference. Both biomarkers indicate that smoking outside the house limits ETS exposure of children but does not eliminate it.     

Correlating atmospheric and biological markers in studies of secondhand tobacco smoke exposure and dose in children and adults

OBJECTIVE: We sought to directly compare secondhand smoke (SHS) atmospheric markers to each other and to SHS dosimetric biomarkers, permitting intercomparison of clinical and atmospheric studies. METHODS: We used atmospheric and pharmacokinetic (PK) models for the quantitative estimation of SHS exposure and dose for infants, children, and adults, based on building smoker density and air exchange rate, and from exposure duration, default PK parameters, and respiration rates. RESULTS: We estimate the SHS serum cotinine doses for the typical and most-exposed individuals in the U.S. population; predictions compare well to measurements on a national probability sample. Using default respiration rates, we estimate serum cotinine dose from SHS nicotine exposure for 40 adults exposed to SHS in an environmental chamber; predictions agreed with observations. We correlate urine cotinine and hair nicotine levels for 127 infants exposed to parental smoking, and estimate corresponding atmospheric nicotine exposure via PK modeling. CONCLUSIONS: Our "Rosetta Stone" Equations allow the SHS atmospheric markers, respirable particles, nicotine, and carbon monoxide, to be related to the SHS biomarkers, cotinine in blood, urine, and saliva and nicotine in hair, permitting intercomparison of clinical and atmospheric studies of SHS for the first time.