Determination of chlormequat and mepiquat in foods by liquid chromatography/mass spectrometry or liquid chromatography/tandem mass spectrometry: interlaboratory study

Interlaboratory validation studies have been performed on 2 methods for the determination of chlormequat (CLQ) and mepiquat (MPQ). Both methods used identical extraction procedures and stable isotope internal standardization but differed in the use of liquid chromatography/mass spectrometry (LC/MS) or LC/tandem mass spectrometry (LC/MS/MS) for the determination, the amount of internal standard used, and the expected limit of detection. After addition of deuterated internal standards, CLQ and MPQ were extracted with methanol-water and determined by LC//MS or LC/MS/MS with positive electrospray ionization. Eight European laboratories participated in the LC/MS method study, analyzing mushroom, pear, wheat flour, and fruit puree with residues of CLQ in the range 0.040-1.19 mg/kg and of MPQ in the range 0.041-0.39 mg/kg. For CLQ, the Horwitz ratio (HoRat) values for individual test materials/levels were in the range 0.85-1.13 with a mean of 1.00, showing good method performance. For MPQ, the Ho values for mushroom, pear (both levels), and wheat flour were in the range 0.83-0.94, again indicating good method performance. For the determination of MPQ in infant food (fruit puree) at 0.041 mg/kg, the Ho was 1.7 when a value of 0 reported by one participant was excluded. In the LC/MS/MS study, in which 11 laboratories participated, a separate sample set was analyzed with residues of CLQ in the range 0.007-1.03 mg/kg and of MPQ in the range 0.008-0.72 mg/kg. Ho values for CLQ were in the range 0.27-1.36 and for MPQ in the range 0.51-2.10, all corresponding to acceptable method performance.     

快速溶剂萃取-气相色谱-串联质谱法分析海洋沉积物中16种多环芳烃

建立了快速溶剂萃取（ASE）-气相色谱-串联质谱（GC-MS/MS）分析海洋沉积物中16种多环芳烃（PAHs）的分析方法。样品由正己烷-丙酮（1∶1,v/v）溶液萃取,经无水硫酸钠脱水、氮吹浓缩后,采用硅胶固相萃取小柱进行净化,然后经HP-5MS色谱柱（30 m×0.25 mm×0.25 μm）分离,在电子轰击电离源下以多反应监测（MRM）模式进行检测,内标法定量。分析结果表明,16种PAHs在0.01~1.00 mg/L范围内线性关系良好,相关系数（R）大于0.997;目标物的加标回收率为75.8%~97.8%;日内与日间精密度（RSD）均小于10%。当取样量为20.0 g时,16种PAHs的方法检出限为0.048~0.234 μg/kg。该法快速、准确、稳定,能够满足海洋沉积物中痕量PAHs的测定。     

Liquid chromatography/negative ion atmospheric pressure photoionization mass spectrometry: a highly sensitive method for the analysis of organic explosives

Gas chromatography/mass spectrometry (GC/MS) is applied to the analysis of volatile and thermally stable compounds, while liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI‐MS) and liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS) are preferred for the analysis of compounds with solution acid‐base chemistry. Because organic explosives are compounds with low polarity and some of them are thermally labile, they have not been very well analyzed by GC/MS, LC/APCI‐MS and LC/ESI‐MS. Herein, we demonstrate liquid chromatography/negative ion atmospheric pressure photoionization mass spectrometry (LC/NI‐APPI‐MS) as a novel and highly sensitive method for their analysis. Using LC/NI‐APPI‐MS, limits of quantification (LOQs) of nitroaromatics and nitramines down to the middle pg range have been achieved in full MS scan mode, which are approximately one order to two orders magnitude lower than those previously reported using GC/MS or LC/APCI‐MS. The calibration dynamic ranges achieved by LC/NI‐APPI‐MS are also wider than those using GC/MS and LC/APCI‐MS. The reproducibility of LC/NI‐APPI‐MS is also very reliable, with the intraday and interday variabilities by coefficient of variation (CV) of 0.2–3.4% and 0.6–1.9% for 2,4,6‐trinitrotoluene (2,4,6‐TNT). Copyright © 2008 John Wiley & Sons, Ltd.     

Anisole, a new dopant for atmospheric pressure photoionization mass spectrometry of low proton affinity, low ionization energy compounds

Atmospheric pressure photoionization (APPI) is a novel method of ionization in liquid chromatography/mass spectrometry (LC/MS). It was originally developed in order to broaden the range of LC/MS ionizable compounds towards less polar compounds that cannot be analyzed by electrospray (ESI) and atmospheric pressure chemical ionization (APCI). Studies done thus far have shown that non-polar compounds that earlier were not ionizable in LC/MS can indeed be ionized by the use of APPI. However, the best ionization efficiency for low polarity samples has been achieved with low proton affinity (PA) solvents that are not suitable in reversed-phase LC (RP-LC). Here it is demonstrated that the signals for analytes with low proton affinities in acetonitrile can be increased 100-fold by using anisole as the dopant for APPI, which takes the sensitivity to the same level achieved in the analysis of high PA analytes.     

QuEChERS/气相色谱质谱法同时测定膨化食品中多环芳烃类物质

建立了同时测定膨化食品中12种多环芳烃(PAHs)的Qu EChERS/气相色谱三重四极杆质谱(GC-MS/MS)的方法。称取2 g(精确至0.01 g)样品,加入10 mL正己烷提取15 min,采用500 mg乙二胺-N-丙基硅烷化硅胶(PAS)和500 mg C18粉末进行净化,净化后进行GC-MS/MS分析测定。实验结果表明,12种多环芳烃在气相色谱中的分离度良好,在1~1000 ng/mL的浓度范围内线性关系良好。加标回收率测定范围为49%~130%,RSD在1.0%~10%(n=7)之间。检出限在0.20~0.66μg/kg之间,定量限在0.66~2.20μg/kg之间。该方法数据可靠、操作简单快捷、灵敏度高,适合大批量样品中PAHs的测定。     

Selective immunoclean-up followed by liquid or gas chromatography for the monitoring of polycyclic aromatic hydrocarbons in urban waste water and sewage sludges used for soil amendment

A selective clean-up procedure using immunoaffinity solid-phase extraction was applied for the trace-level determination of polycyclic aromatic hydrocarbons (PAHs) in urban waste water and sewage sludges used for soil amendment. Anti-pyrene antibodies have been immobilized on a silica-based sorbent and the cross-reactivity of the antibodies towards structurally related compounds were allowed to extract the whole class of priority PAHs. The selectivity of the antibodies provided clean extracts from sludges and, therefore, the identification and quantification were shown to be easier using either liquid chromatography (LC) with UV diode array and fluorescence detection in series or gas chromatography-mass spectrometry (GC-MS), although some loss of up to 50% was observed for the clean-up. The identification of the PAHs by matching of UV and MS spectra was greatly improved. The procedure, including immunoclean-up and LC coupled to diode array and fluorescence detection, was validated using certified reference materials with native PAHs of concentrations in the range of 0.57-2.16 mg/kg (dry sludges).     

Trace analysis of dithiocarbamate fungicide residues on fruits and vegetables by hydrophilic interaction liquid chromatography/tandem mass spectrometry

The simultaneous determination of dithiocarbamate (DTC) fungicide residues on fruits and vegetables was performed by liquid chromatography (LC) on a ZIC-pHILIC column coupled to tandem mass spectrometry (MS/MS). For each DTC subclass, i.e. dimethyldithiocarbamates (DMDs), ethylenebis(dithiocarbamates) (EBDs), and propylenebis(dithiocarbamates) (PBDs), the limits of detection and quantification were approximately 0.001 and 0.005 mg kg(-1), respectively. Recoveries from tomatoes, spiked in the range of 0.05-1 mg kg(-1), averaged between 97 and 101%. Several fruits and vegetables from a local market and different countries of origin (apples, pears, grapes, cherry tomatoes, cocktail tomatoes, cucumbers, tomatoes, tamarillos, papaya, and broccoli) were analyzed by LC/MS/MS, LC/MS, and by the routine CS(2) method. In general, the results obtained by both LC/MS and LC/MS/MS were in good agreement with those obtained by the CS(2) method except for the false positive CS(2) results for broccoli and papaya. The results demonstrate that both LC/MS and LC/MS/MS can be used for routine analyses of DTC residues, whereas LC/MS/MS is more sensitive and selective than LC/MS.     

Amnesic shellfish poisoning toxins in shellfish: estimation of uncertainty of measurement for a liquid chromatography/tandem mass spectrometry method

A liquid chromatography/mass spectrometry (LC/MS) method for amnesic shellfish poisoning toxins in shellfish was developed and validated. Tissue homogenate (4 g) was extracted with 16 mL methanol-water (1 + 1, v/v). Dilution into acetonitrile-water (1 + 9, v/v) was followed by C18 solid-phase extraction cleanup. Domoic acid (DA) and epi-domoic acid were determined by LC/MS/MS with electrospray ionization and multiple reaction monitoring. External calibration was performed with dilutions of a certified reference standard. Advantages of this method include speed, lower detection limits, and a very high degree of specificity. The LC/MS response was highly linear, and there were no significant interferences to the determination of DA. Formal method validation was performed on 4 shellfish species. Fortification studies gave recoveries (mean +/- SD; n = 24) of 93 +/- 14% at 1 mg/kg, and 93.3 +/- 7.6% at 20 mg/kg over all the species. Analysis of a mussel certified reference material showed the bias as < 5%. The limits of detection and quantitation were 0.15 and 0.5 mg/kg, respectively. Routine application of the method over 4 months gave a recovery for the QC sample (1 mg/kg fortified blank mussel homogenate) run with each batch of 88.9 +/- 5.5% (mean +/- SD; n = 37). The total uncertainty of measurement results were estimated as 0.12 (12%) at 0.25-5 mg/kg and 0.079 (7.9%) at 5-50 mg/kg. The major contribution to the uncertainty was the repeatability of the LC/MS determination, probably arising from subtle matrix effects.     

Multifactorial optimization approach for the determination of polycyclic aromatic hydrocarbons in river sediments by gas chromatography-quadrupole ion trap selected ion storage mass spectrometry

A procedure for the determination of very low polycyclic aromatic hydrocarbons (PAHs) concentrations in sediment samples has been developed by gas chromatography-quadrupole ion trap mass spectrometry (GC-QIT MS) after extraction with dichloromethane and purification by using silica gel cleanup. Identification and quantification of analytes were based on the selected ion storage (SIS) strategy using deuterated PAHs as internal standards. In order to search out the main factors affecting the SIS mass spectrometry efficiency, four MS parameters, including target total ion count (TTIC), waveform amplitude (WA), transfer line (XLT) and ion trap temperatures (ITT) were subjected to a complete multifactorial design. The most relevant parameters obtained (TTIC and WA) were optimized by a rotatable and orthogonal composite design. Optimum values for these parameters were selected for the development of the method involving PAH determination in sediment samples. The optimized method exhibited a range of 111-760% higher signal-to-noise (S/N) ratios for PAHs in comparison with the method operated by the default conditions, demonstrating that the multifactorial optimization contributed to substantially improve the sensitivity of the GC-QIT MS determination. The accuracy of the method was verified by analyzing NWRI EC-3 certified reference material (Lake Ontario sediment). The selectivity, sensitivity (limits of quantification were in the range of 0.02-11.0 ng g(-1)), accuracy (recoveries >or=77%) and precision (RSD相似文献   

Preparation and certification of two freshwater sediments certified reference materials for polycyclic aromatic hydrocarbons

Two freshwater sediments certified reference materials (CRMs) for 16 polycyclic aromatic hydrocarbons (PAHs) have been developed by the Institute for Environmental Reference Materials (IERM) of Ministry of Environmental Protection (MEP) in China. The methodology for preparing the CRMs of PAHs in sediments is described in this paper. The collected natural sediment samples were air-dried, ground, homogenised, packed, sterilised and tested on stability and homogeneity. Homogeneity results showed that the between-unit variation was confirmed to be below 4.5% for each compound. Stability was assessed after storage of samples for 16 months at temperature less than 30°C and in shade. The certification of the natural sediment matrix CRMs for PAHs was based on the agreement of results using different analytical techniques including gas chromatography/mass spectrometry (GC/MS) and reversed-phase liquid chromatography (LC) by no less than eight collaborating laboratories including IERM. Results of the homogeneity showed that the calculated u_bb′ was 0.9–2.5% for environmental river standard-4 (ERS-4) and 9–2.3% for environmental lake standard-1 (ELS-1), whereas stability results of total 16 PAHs indicated that the calculated u_rel,lts was 4.2% for ERS-4 and 2.2% for ELS-1. Certified values of 16 PAHs in ERS-4 varied from 8.5 to 167 μg/kg and ranged from 0.036 to 2.8 mg/kg in ELS-1.The good comparability, together with the independent confirmation of the assigned mass fraction by using different methods, confirmed that the CRMs are suitable for the method validation and quality control in soil or sediments analysis.     

Determination of polychlorinated biphenyls by liquid chromatography–atmospheric pressure photoionization–mass spectrometry

This study presents the atmospheric pressure photoionization (APPI) of high‐chlorinated (five or more chlorine atoms) polychlorinated biphenyls (PCBs) using toluene as dopant, after liquid chromatographic separation. Mass spectra of PCB 101, 118, 138, 153, 180, 199, 206 and 209 were recorded by using liquid chromatography‐APPI‐tandem mass spectrometry (LC‐APPI‐MS/MS) in negative ion full scan mode. Intense peaks appeared at m/z that correspond to [M ? Cl + O]^? ions, where M is the analyte molecule. Furthermore, a detailed strategy, which includes designs of experiments, for the development and optimization of LC‐APPI‐MS/MS methods is described. Following this strategy, a sensitive and accurate method with low instrumental limits of detection, ranging from 0.29 pg for PCB 209 to 8.3 pg for PCB 101 on column, was developed. For the separation of the analytes, a Waters XSELECT HSS T3 (100 mm × 2.1 mm, 2.5 µm) column was used with methanol/water as elution system. This method was applied for the determination of the above PCBs in water samples (surface water, tap water and treated wastewater). For the extraction of PCBs from water samples, a simple liquid–liquid extraction with dichloromethane was used. Method limits of quantification, ranged from 4.8 ng l^?1, for PCB 199, to 9.4 ng l^?1, for PCB 180, and the recoveries ranged from 73%, for PCB 101, to 96%, for PCB 199. The estimated analytical figures were appropriate for trace analysis of high‐chlorinated PCBs in real samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparison of dopants for charge exchange ionization of nonpolar polycyclic aromatic hydrocarbons with reversed-phase LC-APPI-MS

Atmospheric pressure photoionization (APPI) is capable of ionizing nonpolar compounds in LC/MS, through charge exchange reactions following photoionization of a dopant. Recently, several novel dopants-chlorobenzene, bromobenzene, 2,4-difluoroanisole, and 3-(trifluoromethyl)anisole-have been identified as having properties making them well-suited to serve as dopants for charge exchange ionization under reversed-phase LC conditions. Here, we report the results of experiments comparing their effectiveness to that of established dopants-toluene, anisole, and a toluene/anisole mixture, for the charge exchange ionization of model nonpolar compounds-the 16 polycyclic aromatic hydrocarbons (PAHs) identified by the US EPA as priority pollutants-when using a conventional reversed-phase LC method. Chloro- and bromobenzene were found to be much more effective than toluene for all the PAHs, due to the relatively low reactivity of their photoions with the solvent. Their overall performance was also better than that of anisole, due to anisole's ineffectiveness toward higher-IE compounds. Further, the experiments revealed that anisole's performance for higher-IE compounds can be dramatically improved by introducing it as a dilute solution in toluene, rather than neat. The two fluoroanisoles provided the highest overall sensitivity, by a slim margin, when introduced as dilute solutions in either chloro- or bromobenzene.     

Fully automated trace level determination of parent and alkylated PAHs in environmental waters by online SPE-LC-APPI-MS/MS

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous compounds that enter the environment from natural and anthropogenic sources, often used as markers to determine the extent, fate, and potential effects on natural resources after a crude oil accidental release. Gas chromatography-mass spectrometry (GC-MS) after liquid–liquid extraction (LLE+GC-MS) has been extensively used to isolate and quantify both parent and alkylated PAHs. However, it requires labor-intensive extraction and cleanup steps and generates large amounts of toxic solvent waste. Therefore, there is a clear need for greener, faster techniques with enough reproducibility and sensitivity to quantify many PAHs in large numbers of water samples in a short period of time. This study combines online solid-phase extraction followed by liquid chromatography (LC) separation with dopant-assisted atmospheric pressure photoionization (APPI) and tandem MS detection, to provide a one-step protocol that detects PAHs at low nanograms per liter with almost no sample preparation and with a significantly lower consumption of toxic halogenated solvents. Water samples were amended with methanol, fortified with isotopically labeled PAHs, and loaded onto an online SPE column, using a large-volume sample loop with an auxiliary LC pump for sample preconcentration and salt removal. The loaded SPE column was connected to an UPLC pump and analytes were backflushed to a Thermo Hypersil Green PAH analytical column where a 20-min gradient separation was performed at a variable flow rate. Detection was performed by a triple-quadrupole MS equipped with a gas-phase dopant delivery system, using 1.50 mL of chlorobenzene dopant per run. In contrast, LLE+GC-MS typically use 150 mL of organic solvents per sample, and methylene chloride is preferred because of its low boiling point. However, this solvent has a higher environmental persistence than chlorobenzene and is considered a carcinogen. The automated system is capable of performing injection, online SPE, inorganic species removal, LC separation, and MS/MS detection in 28 min. Selective reaction monitoring was used to detect 28 parent PAHs and 15 families of alkylated PAHs. The methodology is comparable to traditional GC-MS and was tested with surface seawater, rainwater runoff, and a wastewater treatment plant effluent. Positive detections above reporting limits are described. The virtual absence of sample preparation could be particularly advantageous for real-time monitoring of discharge events that introduce PAHs into environmental compartments, such as accidental releases of petroleum derivates and other human-related events. This work covers optimization of APPI detection and SPE extraction efficiency, a comparison with LLE+GC-MS in terms of sensitivity and chromatographic resolution, and examples of environmental applications.     

大气压光电离技术及其应用研究进展

大气压光电离离子源(APPI)是一种新兴的用于液质联用的软电离离子源,它是利用光化学作用将气相中样品电离的离子化技术,该技术促进了质谱技术对弱极性化合物的分析检测。介绍了液相色谱–质谱/质谱联用技术中大气压光电离的基本原理、应用优点,综述了其在定性、定量分析检测中的应用。     

Determination of benzoylurea insecticides in food by pressurized liquid extraction and LC‐MS

A method based on pressurized liquid extraction and LC‐MS/MS has been developed for determining nine benzoylureas (BUs) in fruit, vegetable, cereals, and animal products. Samples (5 g) were homogenized with diatomaceous earth and extracted in a 22 mL cell with 22 mL of ethyl acetate at 80°C and 1500 psi. After solvent concentration and exchange to methanol, BUs were analyzed by LC‐MS/MS using an IT mass analyzer, which achieved several transitions of precursor ions that increase selectivity providing identification. LOQs were between 0.002 and 0.01 mg/kg, which are equal or lower than maximum residue limits established by the Codex Alimentarius. Excellent linearity was achieved over a range of concentrations from 0.01 to 1 mg/kg with correlation coefficients 0.995–0.999 (n=7). Validation of the total method was performed by analyzing in quintuplicate seven different commodities (milk, eggs, meat, rice, lettuce, avocado, and lemon) at three concentration levels (0.01, 0.1, and 1 mg/kg). The recoveries ranged from 58 to 97% and the RSDs from 5 to 19% depending on the compound and the commodity. The combination of pressurized liquid extraction with LC‐MS/MS provides a sensitive and selective method for the determination of BUs in food.     

Liquid chromatography/atmospheric pressure photoionization tandem mass spectrometry for analysis of Dechloranes

Liquid chromatography/atmospheric pressure photoionization tandem mass spectrometry (LC/APPI-MS/MS) was investigated as an instrumental method for the analysis of the halogenated norbornene flame retardants, Mirex, Dechloranes 602, 603, 604, and Dechlorane Plus (DP). The LC separation was optimized by screening a variety of stationary and mobile phases, resulting in a short LC separation time of 5 min. Different atmospheric pressure ionization approaches were examined including electrospray ionization, atmospheric pressure chemical ionization, and APPI, each with and without post-column addition. APPI without post-column addition was chosen for providing the best ionization response. The optimized LC/APPI-MS/MS approach resulted in instrument detection limits ranging between 25 and 50 pg. Good linearity was also achieved (up to 25.0 ng/μL; R >0.999). The method was applied to extracts of environmental samples including surface water, fish and sediments for screening purposes, and the results agreed well with those obtained by gas chromatography/mass spectrometry.     

Development of liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry for analysis of halogenated flame retardants in wastewater

Until recently, atmospheric pressure photoionization (APPI) has typically been used for the determination of non-polar halogenated flame retardants (HFRs) by liquid chromatography (LC) tandem mass spectrometry. In this study, we demonstrated the feasibility of utilizing liquid chromatography atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (LC-APCI-MS/MS) for analysis of 38 HFRs. This developed method offered three advantages: simplicity, rapidity, and high sensitivity. Compared with APPI, APCI does not require a UV lamp and a dopant reagent to assist atmospheric pressure ionization. All the isomers and the isobaric compounds were well resolved within 14-min LC separation time. Excellent instrument detection limits (6.1 pg on average with 2.0 μL injection) were observed. The APCI mechanism was also investigated. The method developed has been applied to the screening of wastewater samples for screening purpose, with concentrations determined by LC-APCI-MS/MS agreeing with data obtained via gas chromatography high resolution mass spectrometry.

Development and validation of a liquid chromatographic/ tandem mass spectrometric method for determination of chlortetracycline, oxytetracycline, tetracycline, and doxycycline in animal feeds

A selective and accurate LC/MS/MS method for the simultaneous determination of chlortetracycline (CTC), oxytetracycline (OTC), tetracycline (TC), and doxycycline (DC) in animal feeds was developed. Samples were extracted with Na2EDTA-McIlvaine buffer and further purified with Oasis HLB SPE columns. The purified extract was separated on an Xbridge C18 column and detected by LC/MS/MS with positive electrospray ionization in the multiple reaction monitoring mode. This method provided average recoveries of 80.9 to 119.5%, with CVs of 1.7 to 9.8% in the range of 0.5 to 50 mg/kg CTC, OTC, TC, and DC in feeds, except the average recovery of CTC was 76.0%, with a CV of 14.6% in pig feed spiked with 0.5 mg/kg CTC. The linear ranges for the four TCs determined by LC/MS/MS ranged from 0.005 to 2.5 microg/mL with a linear correlation coefficient (R2) >0.99. The LOD and LOQ for CTC, OTC, TC, and DC in pig and poultry feeds ranged from 0.003 to 0.02 and 0.01 to 0.05 microg/g, respectively. The method was successfully applied for the analysis of 30 real feed samples, and no illegal use was detected.     

Applicability of gradient liquid chromatography with tandem mass spectrometry to the simultaneous screening for about 100 pesticides in crops

A method was developed for screening crops for a range of pesticide residues by liquid chromatography/tandem mass spectrometry (LC/MS/MS). A complete set of LC, electrospray ionization (ESI), and tandem MS acquisition parameters was established for the determination of 108 analytes; these parameters were used for the simultaneous acquisition of 98 analytes in the positive ESI mode and 10 analytes in an additional MS/MS method in the negative ESI mode. The entire procedure involves extraction of residues with methanol-water and partition into dichloromethane. The utility of the method is demonstrated by the analysis of crops of 5 matrix types (water-containing, acidic, dry, sugar-containing, and fatty). Of 108 pesticides/metabolites tested, 104 showed sufficient stability in most matrixes for determination by LC/MS/MS. These analytes belong to 20 chemical classes, which demonstrate the general applicability of the method for multiclass analysis. By using matrix-matched standards, 67 compounds could be determined in most matrixes with recoveries of 70-120% and a relative standard deviation of < or = 25% at the 0.01 mg/kg level.     

Comparison of analyses of urinary 8-hydroxy-2'-deoxyguanosine by isotope-dilution liquid chromatography with electrospray tandem mass spectrometry and by enzyme-linked immunosorbent assay

A highly sensitive and selective method, using isotope-dilution liquid chromatography with tandem mass spectrometry (LC/MS/MS), for quantification of urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), an important biomarker of oxidative stress, was developed and compared with a method using an enzyme-linked immunosorbent assay (ELISA). The synthesis of (15)N(5)-8-OHdG is described. In this study, 140 urine samples were collected from workers in a coke oven plant, including samples from 49 control workers and 91 workers who had been occupationally exposed to polyaromatic hydrocarbons (PAHs). The major urinary metabolite of PAHs, 1-hydroxypyrene (1-OHP), was measured for the exposed workers. Results from the present study showed a significant correlation between these two measurements for determination of 8-OHdG (p < 0.05, r(2) = 0.70). However, only the LC/MS/MS measurements of urinary levels of 8-OHdG showed a significant difference between the exposed and the control subjects (p < 0.05). The ELISA method failed to demonstrate this difference. Furthermore, only by using the LC/MS/MS method was a significant correlation observed between the urinary levels of 1-OHP and 8-OHdG. These findings suggest that a highly specific and sensitive analytical method such as isotope-dilution LC/MS/MS is extremely important and necessary for accurate measurement and a comprehensive study of oxidative stress in human subjects.