超高效液相色谱－串联质谱联用检测血清胆汁酸谱方法的建立及初步临床应用

目的 建立超高效液相色谱-串联质谱(UPLC-MS/MS)联用方法检测血清中胆汁酸表达的方法,并初步探讨胆汁酸亚型与肥胖的关系.方法 采用固相萃取方法处理血清标本,经UPLCHSS T3色谱柱梯度洗脱后,用负离子多反应监测模式对血清中的胆汁酸组分进行质谱检测,并根据中国国家食品药品监督管理局(SFDA)的指导原则对该方法的特异性、线性范围、灵敏度、不精密度和相对回收率进行性能验证.利用UPLC-MS/MS方法检测10例单纯性肥胖者和10名健康对照者血清胆汁酸浓度,并采用秩和检验进行统计分析.结果 UPLC-MS/MS方法可同时检测人血清中14种胆汁酸亚组分,在10～20 000 nmol/L范围内线性相关良好.其对不同亚组分的最低检出限(LOD)为0.02～7.90 nmol/L,最低定量限(LOQ)为0.07～44.20 nmol/L;其日内不精密度为0.35％～12.41％;日间不精密度为1.11％ ～13.04％;相对回收率为89.8％～114.6％.初步临床应用显示,单纯性肥胖者及健康对照者血清胆汁酸谱色谱图有一定差异,单纯性肥胖者血清中游离胆汁酸和结合胆汁酸浓度分别为0.49(0.45 ～1.90)、1.44(0.84～3.72) μmol/L,均低于健康对照组的0.98(0.53～3.06)、1.99(0.67 ～2.88) μmol/L,但差异均无统计学意义(Z=-0.958、-0.801,P均＞0.05).结论 建立的UPLC-MS/MS方法可有效分离并定量检测血清中各种胆汁酸亚型,其线性范围广、灵敏度高、精密度好,可用于临床标本游离胆汁酸和结合胆汁酸的分析测定.     

串联质谱技术检测孕妇胆汁酸谱的性能评估及其参考区间建立

目的对基于串联质谱(LC-MS/MS)技术检测胆汁酸谱的方法进行性能评估并建立适合于南京地区孕妇的15种胆汁酸亚型参考区间。方法采用LC-MS/MS技术检测孕中期孕妇血清15种胆汁酸亚型,根据美国临床和实验室标准协会(CLSI)C62-A和美国食品药品监督管理局的生物分析方法验证标准,从定量检出限、准确度、精密度、线性、携带污染率等几个方面进行性能评估;根据CLSI EP28-A3c推荐的方法和程序建立南京地区孕妇孕中期15种胆汁酸亚型的参考区间,用小样本验证法对新建立的20例样本参考区间进行验证。结果 LC-MS/MS技术可同时检测孕妇血清中15种胆汁酸亚组分,最低定量限为标准曲线最低浓度点(S1)浓度,最低检出限为1/4 S1;线性:各种亚型均呈现良好的线性关系,r~2均大于0.99;准确度:各种亚型加标回收试验回收率均为80%～120%,偏倚15%;批内精密度和批间精密度变异系数均小于15%;携带污染率:小于最低定量限的25%;孕中期孕妇参考区间:以石胆酸为例,新的参考区间为0.07～13.46 ng/mL,95%的验证结果落于新的参考区间内。结论 LC-MC/MS技术可以精准、快速地检测胆汁酸谱15种亚型,评估结果符合各项性能验证要求;新建立的胆汁酸谱15种亚型的参考区间适合于南京地区孕中期孕妇。     

流动注射--时间分辨荧光联用检测CEA

目的 建立流动注射与时间分辨荧光分析联用技术测定血清中癌胚抗原(CEA)的免疫分析方法(FIIA)。方法 流动注射免疫反应柱的制备；快速流动注射分析法的探索；免疫反应最佳条件的选择；标准曲线的绘制：回收试验。结果 方法的检测线性范围在0～100ng／ml，回收率为96．5％～102％。结论 该文所建立的方法具有快速、准确且能定量的特点，具有较好的应用前景。     

除草剂2,4-D丁酯的GC/MS检测及其在中毒患者诊治中的应用

目的建立除草剂2,4-D丁酯的气相色谱-质谱（GC／MS）检测方法,用于临床中毒患者的检测。方法以乙酸乙酯提取血液中2,4D丁酯,采用GC／MS检测。定性采用全扫描法（SCAN）,定量采用选择离子法（SIM）,离子选择m／z175、185、276。结果2,4-D丁酯在10～1500ng／ml成线性,方程为Y=10．565X-1203．5,R=0．9941,标加1000ng／ml浓度的2,4-D丁酯于血液,平均回收率为94．6％,RSD=7．1％;2,4-D丁酯最低检出浓度为3ng／ml（S1M）,信噪比S／N=3;日内精密度RSD为4．1％,日间精密度RSD为5．8％。结论所建方法灵敏、快速、线性范围好,可用于2,4-D丁酯中毒患者的检测。     

血清肌红蛋白光激化学发光免疫测定法的建立

目的采用光激化学发光免疫测定技术（LICI）建立定量检测肌红蛋白（Mb）的方法。方法使用针对Mb不同表位的2株单克隆抗体，一株包被发光微粒，另一株进行生物素化，与包被有链霉亲合素的感光微粒共同构成检测试剂，优化检测条件并评价检测性能。结果该方法的灵敏度为1．36ng／ml，在33．8～1521．0ng／ml的范围内线性良好。批内和日间变异系数分别为3．05％～4．41％和5．80％～6．56％。在血红蛋白浓度〈2．6g／L，总胆红素浓度〈342μmol／L、三酰甘油浓度〈11．3mmol／L时的干扰率无临床意义。与Roche Elecsys电化学发光法有较好的相关性（r=0．9971）。结论血清MbLICI的建立有助于进一步对检测试剂盒的研制。     

血清降钙素原测定在感染性疾病中的诊断价值

目的探讨血清降钙素原（PCT）检测在感染性疾病中的诊断价值。方法对342例各种感染性疾病的儿童患者采用半定量固相免疫测定法测定患者血清PCT水平，PCT水平分别为〈O．5、0．5～2．0、～10和〉10ng／ml4个等级。结果若以血清PCT≥0．5ng／ml为阳性周值，则血清PCT检测对细菌感染诊断的敏感性为75％、特异性为70．2％，阳性预测值为45．0％，阴性预测值为89．6％。结论血清PCT是细菌感染的重要指标，其诊断价值明显优于外周血白细胞计数和分类；血清PCT水平高低可作为是否使用抗菌药物的参考依据。     

固相萃取-反相高效液相色谱法同时测定血清中3种抗癫痫药物

目的应用固相萃取-反相高效液相色谱法测定血清中抗癫痫药物。方法用Elut Bond C18小柱预处理样品，甲醇为洗脱剂；采用Waters Nova RC18柱，流动相为甲醇：水-45：55；检测波长230nm，流速1ml／min，内标(苯乙酮)法定量。结果苯巴比妥、苯妥英、卡马西平的浓度线性范围分别为4．1～62．6μg／ml(r=0．9968)、3．9～60．1μg／ml(r=0．9971)和1．6～21．2μg／ml(r=0．9945)，最低检测限分别为0．05μg／ml、0．05μg／ml和0．01μg／ml；相对回收率分别为90．6％～103．6％、93．4％～118．3％和91．2％～101．1％；批内变异系数均小于6．0％，批间变异系数均小于8．0％。结论固相萃取-反相高效液相色谱法简便、灵敏、准确、稳定，适合于以上3种抗癫痫治疗药物的同时监测。     

时间分辨荧光免疫分析法乙型肝炎血清标志物分析灵敏度的建立与分析

[目的]乙型肝炎表面抗原和表面抗体定量测定试剂盒。[方法]时间分辨荧光免疫分析法的分析灵敏度进行建立与分析，以便更好的指导临床诊断和进行流行病学研究。[结果]通过实验研究，乙型肝炎病毒表面抗原定量检测试剂盒和表面抗体定量检测试剂盒的检测低限分别为0．0356ng／ml和0．548mIU／ml，生物检测限分别为0．200ng／ml和5．00mIU／ml，功能灵敏度分别为0．200ng／ml和5．00mIU／ml，线性范围分别为0．200ng／ml-300ng／ml和5．00mIU／ml～1200mIU／ml，并达到中国药品生物制品检定所检定要求。[结论]时间分辨荧光免疫分析法乙型肝炎病毒定量检测试剂盒具有灵敏度高，线性范围宽，特异性强的特点，已能有效应用于临床体外诊断和流行病学研究。     

茜素红S直接光度法测定血清白蛋白的方法学评价

目的：建立准确、灵敏、特异、微量的茜素红S(ARS)光度法测定血清白蛋白。方法：建立检测血清白蛋白含量的茜素红S法最适条件并进行方法学系列评价实验。结果：该法线性范围为0～75g／L，手工法批内和批间变异系数分别为2．1％，3．4％；回收率97．2％～102．0％。自动分析法批内和批间变异系数分别为1．9％，2．5％；回收率97．7％～103．2％。与BCG法比较有良好的相关性，相关系数r=0．9913，相关方程y=0．9783x 2．043。γ-球蛋白、胃蛋白酶、溶茵酶、胰蛋白酶对测定基本无干扰。血清甘油三酯浓度3．0mmol／L以下，血红蛋白浓度2．5g／L以下，胆红素浓度342μmol／L以下对测定无影响。结论：茜素红S测定白蛋白方法血清微量，操作简单，线性范围宽，重复性好，准确度高，适用于手工和自动生化分析仪检测。     

时间分辨荧光免疫法和电化学发光法检测AFP的比较

目的对时间分辨荧光免疫分析法（TRFIA法）国产试剂盒与电化学发光免疫分析法（ECLIA法）检测血清AFP作比较分析。了解两种方法特点。方法分别采用国产TRFIA法试剂盒与ECLIA法检测正常人50例、肝癌患者40例血清AFP的含量;用高、中、低3种浓度AFP标准品采用两种方法进行批内测定,n=10,观察重复性,所得结果计算CV值：将高值标准品血清（250ng／ml）按1：1比例加入每份正常对照组血清中,肝癌组病人血清与生理盐水按1：1稀释后再按1：1比例加入高值标准品血清（250ng／ml）,每份检测2次（TRFIA法）取平均值,计算回收率。结果肝癌组AFP值（TRFIA法：558±319．10ng／ml;ECLIA法：552．31±302．67ng／ml）明显高于正常组（TRFIA法：6．51±5．42ng／ml;ECLIA法：6．32±5．14ng／m）（P〈0．05）,两种方法结果无显著差异（P〉0．05）,其相关系数为0．9150,测定结果呈正相关。高、中、低3种浓度标准品的重复性试验TRFIA法与ECLIA批内变异系数分别低于7．9％和3．1％,后者优于前者,两种方法略有不同,无明显差异（P〉0．05）。回收试验结果,TRFIA法回收率在92．3％～103．9％。结论国产TRFIA法试剂盒和ECLIA法检测AFP相比较具有良好的相关性。灵敏度和稳定性也较好．可应用于临床血清AFP检测。     

串联质谱法测定血清中20种游离氨基酸含量

目的建立串联质谱法(LC-MS/MS)测定血清中20种游离氨基酸含量,为临床血清氨基酸检测提供可靠方法。方法血清样本未经衍生化处理,采用LC-MS/MS法,于电喷雾源(ESI)正离子模式下使用选择反应性离子扫描(SRM)进行定量。结果 20种氨基酸线性相关系数为0.9957～0.9999,日内、日间变异系数分别为0.5%～9.0%和0.6%～12.3%,加样回收率为87.6%～115.5%。样本在常温放置24h,8次冻融循环及-20℃冻存20d等条件下稳定性良好。结论该方法简便、稳定、定量准确,适用于血清氨基酸的临床检测,为氨基酸代谢疾病的临床诊断提供了重要的生化依据。     

同位素稀释超高效液相色谱串联质谱法检测血浆18-羟皮质酮方法的建立及初步临床应用

  目的  建立一种同位素稀释超高效液相色谱串联质谱法(isotope dilution ultra performance liquid chromatography tandem mass spectrometry, ID-UPLC-MS/MS)检测血浆18-羟皮质酮(18-Hydroxycorticosterone,18-OHB)的方法。  方法  取血浆标本或标准溶液200 μL置于离心管中,然后加入同位素氚标记的18-OHB为内标,用甲醇沉淀血浆蛋白,离心后取上清液,用Prime HLB Elution 96孔SPE板进行萃取,收集洗脱液,在正离子电喷雾离子化的多离子反应监测模式下检测18-OHB。评价该方法的精密度、加标回收率、定量检测下限、线性和基质效应。2019年11月起招募表观健康志愿者,采用ID-UPLC-MS/MS检测其血浆18-OHB水平,并验证梅奥医学实验中心提供的18-OHB参考区间。  结果  该方法检测血浆18-OHB的分析时间约为3.0 min,检测低、中、高3个水平18-OHB的重复性变异系数和实验室内不精密度分别为2.2%~3.5%和3.7%~5.0%,平均加标回收率为98.1%~101.7%,定量检测下限为0.01 μg/L,在0.1~10 μg/L范围内线性良好(r＞0.990),血浆基质效应为86.83%~119.00%。基于本研究建立的ID-UPLC-MS/MS方法,73名表观健康人群血浆18-OHB第2.5、97.5百分位数分别为0.01、0.60 μg/L,其中66%(48/73)血浆18-OHB水平处于梅奥医学实验中心提供的参考范围外。  结论  本研究建立了一种ID-UPLC-MS/MS测定血浆18-OHB的方法,该方法检测快速、结果准确可靠,性能满足临床需求。     

Rapid screening assay of congenital adrenal hyperplasia by measuring 17 alpha-hydroxyprogesterone with high-performance liquid chromatography/electrospray ionization tandem mass spectrometry from dried blood spots

A rapid, simple, and specific method was developed for the diagnosis of congenital adrenal hyperplasia (CAH) from dried blood spots on newborn screening cards based on high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). The usefulness of 17 alpha-hydroxyprogesterone (17 OH-P) determination on dried filter-paper blood samples from patients with CAH caused by 21-hydroxylase deficiency was evaluated. The LC/MS/MS detection of 17 OH-P was rapid, <4 min. The intra- and interday accuracy and precision of the method were <7%. Our procedure maintained good linearities (R(2) > 0.992) and recovery rate (>83%). We used this new method to directly determine the 17 OH-P levels in dried blood specimens from abnormal children of various ages, with a detection limit of 20 ng/ml (approximately 240 pg), to avoid the time-consuming derivatization steps required by the gas-chromatography/mass spectrometry (GC/MS) method. Four dried filter-paper blood samples of CAH patients (three girls and one boy, 1-14 years old) were all quantified in an LC/MS/MS study and revealed high 17 OH-P levels (>90 ng/ml). After treatment, all of the elevated 17 OH-P levels either decreased or disappeared. Compared with CAH patients, 17 OH-P was nearly undetectable (<20 ng/ml) in the normal infants by LC/MS/MS. This LC/MS/MS assay is not only useful for both diagnosis and monitoring of treatment of CAH in all other age groups, it also can be used as a screening test for CAH infants. In this study, we provided the first data on 17 OH-P in dried blood specimens affected with CAH using HPLC/ESI-MS/MS.     

液相色谱-串联质谱法测定人血浆中二甲双胍的浓度

目的采用液相色谱-串联质谱法测定人血浆中二甲双胍的浓度。方法血浆样品用乙腈(含0.1%甲酸)沉淀蛋白后用二氯甲烷反洗后进行分析。使用Agilent C8(75 mm×4.6 mm,3.5μm)色谱柱。流动相:A泵:5 mmol/L醋酸铵(三乙胺调pH值至7.5),B泵:乙腈。线性梯度洗脱,流速0.4 mL/min。采用电喷雾离子源,多反应离子监测。用于定量分析的离子对二甲双胍为130.2/71.1,内标吗啉胍为172.2/60.2。结果线性范围为50～2 000 ng/mL,最低定量限为50 ng/mL,预处理回收率为81.7%～98.0%,二甲双胍的基质效应<9.97%,日内和日间相对标准偏差均<5.2%。结论液相色谱-串联质谱法快速、简便、灵敏度高,是一种适用于人血浆中药物浓度的测定及药物动力学和生物利用度研究的方法。     

Quantification by liquid chromatography tandem mass spectrometry of mycophenolic acid and its phenol and acyl glucuronide metabolites

BACKGROUND: We developed and validated a rapid and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedure for the quantification of mycophenolic acid (MPA) and its phenol glucuronide (MPAG) and acyl glucuronide (AcMPAG) metabolites. METHODS: We performed protein precipitation on all samples (calibrators, quality controls, and patient samples) and then subjected them to online solid-phase extraction followed by reversed-phase liquid chromatography for 4.0 min. The carboxybutoxy ether of MPA (MPAC) was used as the internal calibrator. The separated compounds (MPA, MPAG, AcMPAG, and MPAC) were detected by electrospray ionization-coupled MS/MS. We compared LC-MS/MS results with results for the same samples obtained with a validated HPLC procedure with an ultraviolet detector. RESULTS: Comparison with the validated HPLC-ultraviolet procedure demonstrated good agreement. The Passing-Bablok regression was y = 0.968x - 0.058 for MPA, y = 1.08x - 1.697 for MPAG, and y = 0.952x + 0.076 for AcMPAG. Assay imprecision showed a CV <10% at 3 concentrations for each compound. The lower limit of quantification was 0.1 mg/L for MPA, 1.0 mg/L for MPAG, and 0.05 mg/L for AcMPAG. The mean analytical recovery was 90%-110%. The assay was linear from 0.1 to 50 mg/L for MPA (r = 0.9987), from 1 to 500 mg/L for MPAG (r = 0.9999), and from 0.05 to 10 mg/L for AcMPAG (r = 0.9988). Quantification of the compounds was not affected by in-source fragmentation or ion suppression. CONCLUSION: The LC-MS/MS assay described here is valid and reliable for the quantification of total MPA, MPAG, and AcMPAG in serum.     

Determination of 17alpha-hydroxyprogesterone in serum by liquid chromatography-tandem mass spectrometry and immunoassay

17Alpha-hydroxyprogesterone (17OHP) is the most important serum marker for congenital adrenal hyperplasia (CAH). 17OHP is usually measured by immunoassay but its detection by mass spectrometry (MS) is a potentially superior method. An LC-MS (liquid chromatography-mass spectrometry) method was developed which utilizes 0.5 ml serum spiked with 6-alpha-methylprednisolone (6-MP) or deuterated 17OHP (d8-IS) as the internal standard. The samples were extracted with ether/ethylacetate, and the extract was evaporated to dryness and analysed by LC-MS/MS operating in the positive mode after separation on a reversed-phase C18 column. The calibration curves for analysis of serum 17OHP exhibited consistent linearity and reproducibility in the range of 5-250 nmol/l. Interassay CVs were 8.5 and 9.2% at mean concentrations of 7.9 and 23 nmol/l, respectively. The detection limit was 1 nmol/l (signal-to-noise ratio=3). The mean recovery of 17OHP added to serum ranged from 76 to 89% and that of internal standards from 75 to 82%. The regression equation for the LC-MS/MS (x) and in-house radioimmunoassay (RIA) (y) methods was: y=0.87x+0.26 (r=0.97; n=100) and for a commercial RIA it was: y=1.32x+0.02 (r=0.97; n=26).     

单纯性肥胖者血清胆汁酸谱研究

目的探讨单纯性肥胖者血清胆汁酸谱的特征,为阐明胆汁酸引发的能量代谢相关机制和探寻新的肥胖生物治疗靶点提供研究依据。方法利用超高效液相色谱-串联质谱(UPLC-MS/MS)联用方法检测体检人群中10名表观正常对照者、10例超重者和10例单纯性肥胖者血清胆汁酸谱的特征。结果表观正常对照组、超重组和单纯性肥胖组血清中14种胆汁酸亚组分均有表达,其中的石胆酸(LCA)、牛磺胆酸(TCA)、牛磺石胆酸(TLCA)和牛磺熊脱氧胆酸(TUDCA)的表达水平最低;14种胆汁酸亚组分在各组间的差异均无统计学意义(P>0.05);单纯性肥胖组的胆汁酸谱中以甘氨鹅脱氧胆酸(GCDCA)和牛磺鹅脱氧胆酸(TCDCA)增高为主;超重组及单纯性肥胖组血清游离型胆汁酸(FBA)和结合胆汁酸(CBA)水平稍低于表观正常对照组,但差异无统计学意义(P>0.05)。结论单纯性肥胖者血清胆汁酸谱中以GCDCA和TCDCA增高为主,但各种胆汁酸亚组分的表达水平与表观正常者相同。     

A simple and reliable bile acid assay in human serum by LC‐MS/MS

BackgroundBile acids, as important signaling molecules and regulatory factors acting on glucose, lipid, and energy metabolism, are always involved in liver, biliary, and intestinal diseases. Development and validation of a simple liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for determination of bile acids is significant for the routine clinical testing.MethodsFifty microlitre of serum was mixed with 10 μl of the internal standard working solution and then 140 μl of methanol for protein precipitation. After centrifuged, the supernatant was directly used for LC‐MS/MS analysis.ResultsGood separation of all bile acid species was achieved. The method was validated with consistent linearity for individual bile acids, good recovery, low carryover, satisfactory sample stability, and analytical specificity against hemolysis, lipemia, and bilirubinemia. The intra‐day and the inter‐day imprecision values were in the range of 1.53%–10.63% and 3.01%–13.98%, respectively. No obvious matrix effect was observed. The reference intervals of bile acids in adults have been established for the clinical testing.ConclusionsThe low sample volume, simple sample preparation, good separation of all species, and satisfying validation results make this LC‐MS/MS approach suitable for usage as a high‐throughput assay in routine clinical laboratories.     

液相色谱-串联质谱法监测血清茶碱浓度及室间质量评价

目的对液相色谱-串联质谱(LC-MS/MS)检测茶碱进行方法学评价,探讨其在茶碱治疗药物监测(TDM)中的应用。方法在血清添加放射性核素内标茶碱-D6,经蛋白沉淀稀释后采用LC-MS/MS测定。以Capcell C18 MGⅢ(100 mm×2.0 mm,5μm)为分析柱进行反相色谱分离;以0.1%甲酸乙腈-0.1%甲酸水[20∶80(v/v)]为流动相,流速为0.3 m L/min;以电喷雾离子化串联四级杆质谱、正离子多反应监测进行定量检测。用建立的方法从2008年起连续参加卫生部临检中心茶碱TDM室间质量评价。结果 LC-MS/MS检测茶碱的线性范围为1~50μg/m L,批内和批间精密度分别为2.26%~6.65%和4.70%~6.84%,准确度分别为94.14%~104.00%。单个样品的监测分析时间为3.5 min。冻融(-30℃室温反复解冻3次)、室温放置24 h、自动进样器放置24 h、长期保存(-30℃放置28 d)的稳定性均良好。LC-MS/MS测定结果与室间质量评价靶值偏差为2.75%,斜率为1.04,相关系数(r2)为0.983。结论该LC-MS/MS采用放射性内标稀释,具有简单、快速、特异性和灵敏度较好的特点,连续6年测定结果符合全国室间质量评价要求,可用于茶碱的临床TDM。     

Clinical-scale high-throughput analysis of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine by isotope-dilution liquid chromatography-tandem mass spectrometry with on-line solid-phase extraction

BACKGROUND: Quantification of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in urine or blood is used to assess and monitor oxidative stress in patients. We describe the use of on-line solid-phase extraction (SPE) and isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) for automated measurement of urinary 8-oxodGuo. METHODS: Automated purification of urine was accomplished with a switching valve and an Inertsil ODS-3 column. After the addition of 15N5-labeled 8-oxodGuo as an internal standard, urine samples were analyzed within 10 min without sample purification. This method was applied to measure urinary 8-oxodGuo in a group of healthy persons (32 regular smokers and 35 nonsmokers). Urinary cotinine was also assayed by an isotope-dilution LC-MS/MS method. RESULTS: The lower limit of detection was 5.7 ng/L on column (2.0 fmol). Inter- and intraday imprecision (CV) was < 5.0%. Mean recovery of 8-oxodGuo in urine was 99%-102%. Mean (SD) urinary concentrations of 8-oxodGuo in smokers [7.26 (3.14) microg/g creatinine] were significantly higher than those in nonsmokers [4.69 (1.70) microg/g creatinine; P < 0.005]. Urinary concentrations of 8-oxodGuo were significantly correlated with concentrations of cotinine in smokers (P < 0.05). CONCLUSIONS: This on-line SPE LC-MS/MS method is sufficiently sensitive, precise, and rapid to provide high-throughput direct analysis of urinary 8-oxodGuo without compromising quality and validation criteria. This method could be applicable for use in daily clinical practice for assessing oxidative stress in patients.