Analysis of N7-(benzo[a]pyrene-6-yl)guanine in urine using two-step solid-phase extraction and isotope dilution with liquid chromatography/tandem mass spectrometry

Analysis of urinary N7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a DNA adduct induced by benzo[a]pyrene, may serve as a risk-associated biomarker for exposure to polyaromatic hydrocarbons (PAHs). In this study a highly sensitive and specific analytical method, incorporating on-line sample preparation coupled with isotope-dilution liquid chromatography and tandem mass spectrometry (LC/MS/MS), was developed to quantitate this adduct in human urine. In order to achieve accurate quantitation, 15N5-labeled BP-6-N7Gua was synthesized to serve as the internal standard, and a two-step solid-phase extraction (SPE) procedure using C8 and SCX cartridges was used for sample cleanup. BP-6-7-N7Gua was analyzed using positive ion LC/MS/MS operated in multiple reaction monitoring (MRM) mode. The [M+H]+ ions at m/z 402 and 407 and the common fragment ion of [M+H]+ at m/z 252 were monitored for quantification. The recovery of this analyte after two-step SPE was 90%, and the limit of detection was 2.5 fmol/mL in 10 mL of urine. This highly specific and sensitive method for BP-6-N7Gua in urine may be applied to assess exposure to PAHs in coke-oven workers for future molecular epidemiology studies on health effects of PAHs.     

Highly sensitive routine method for urinary 3-hydroxybenzo[a]pyrene quantitation using liquid chromatography-fluorescence detection and automated off-line solid phase extraction

Many workers and also the general population are exposed to polycyclic aromatic hydrocarbons (PAHs), and benzo[a]pyrene (BaP) was recently classified as carcinogenic for humans (group 1) by the International Agency for Research on Cancer. Biomonitoring of PAHs exposure is usually performed by urinary 1-hydroxypyrene (1-OHP) analysis. 1-OHP is a metabolite of pyrene, a non-carcinogenic PAH. In this work, we developed a very simple but highly sensitive analytical method of quantifying one urinary metabolite of BaP, 3-hydroxybenzo[a]pyrene (3-OHBaP), to evaluate carcinogenic PAHs exposure. After hydrolysis of 10 mL urine for two hours and concentration by automated off-line solid phase extraction, the sample was injected in a column-switching high-performance liquid chromatography fluorescence detection system. The limit of quantification was 0.2 pmol L(-1) (0.05 ng L(-1)) and the limit of detection was estimated at 0.07 pmol L(-1) (0.02 ng L(-1)). Linearity was established for 3-OHBaP concentrations ranging from 0.4 to 74.5 pmol L(-1) (0.1 to 20 ng L(-1)). Relative within-day standard deviation was less than 3% and relative between-day standard deviation was less than 4%. In non-occupationally exposed subjects, median concentrations for smokers compared with non-smokers were 3.5 times higher for 1-OHP (p<0.001) and 2 times higher for 3-OHBaP (p<0.05). The two urinary biomarkers were correlated in smokers (ρ=0.636; p<0.05; n=10) but not in non-smokers (ρ=0.09; p>0.05; n=21).     

The use of chemical derivatization to enhance liquid chromatography/tandem mass spectrometric determination of 1-hydroxypyrene, a biomarker for polycyclic aromatic hydrocarbons in human urine

This article presents an analytical approach that used chemical derivatization to enhance mass spectrometric (MS) response in electrospray ionization (ESI) mode of 1-hydroxypyrene (1-OHP), a commonly used biomarker to monitor human exposure to polycyclic aromatic hydrocarbons (PAHs). The enhancement successfully enabled the desired detection of 50 pg/mL in human urine. The introduction of an MS-friendly dansyl group to 1-OHP enhanced both ionization efficiency in the ESI source and collision-activated dissociation (CAD) in the collision cell. The response increase was estimated to be at least 200-fold, and enabled the reduction of sample size to only 100 microL. The selective MS detection also facilitated a fast (run time 3 min) liquid chromatography (LC) method which successfully resolved the analyte and interferences. The sample processing procedure included enzymatic hydrolysis of glucuronide and sulfate conjugates, liquid-liquid extraction, derivatization with dansyl chloride and a final liquid-liquid extraction to generate clean extracts for LC/MS/MS analysis. This approach has been validated as sensitive, linear (50-1000 pg/mL), accurate and precise for the quantitation of 1-OHP in human urine. This is the first report of using chemical derivatization to enhance MS/MS detection with fast chromatography in the determination of 1-OHP in human urine.     

Rapid and sensitive quantification of urinary N7-methylguanine by isotope-dilution liquid chromatography/electrospray ionization tandem mass spectrometry with on-line solid-phase extraction

A rapid, highly specific and sensitive isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) method coupled with an on-line solid-phase extraction (SPE) system was developed to measure N7-methylguanine (N7-MeG) in urine. 15N5-Labeled N7-MeG was synthesized to serve as an internal standard, and an on-line SPE cartridge was used for on-line sample cleanup and enrichment. The urine sample can be directly analyzed within 15 min without prior sample purification. The detection limit for this method was estimated as 8.0 pg/mL (4.8 pmol) on-column. This method was further applied to study exposure to methylating agents arising from cigarette smoke. Sixty-seven volunteers were recruited, including 32 regular smokers and 35 nonsmokers. Urinary cotinine, a major metabolite of nicotine, was also determined using an isotope-dilution LC/MS/MS method. The results showed that urinary levels of N7-MeG observed in smokers (4215 +/- 1739 ng/mg creatinine) were significantly (P < 0.01) higher than those in nonsmokers (3035 +/- 720 ng/mg creatinine). It was further noted that the urinary level of N7-MeG was found to be correlated with that of cotinine for smokers, implying that cigarette smoking resulted in increased DNA methylation, followed by depurination and excretion of N7-MeG in urine. As a result of the on-line extraction system, this method is capable of routine high-throughput analysis and accurate quantitation of N7-MeG, and could be a useful tool for health surveillance of methylating agent exposure.     

Molecularly imprinted monolith in-tube solid-phase microextraction coupled with HPLC/UV detection for determination of 8-hydroxy-2′-deoxyguanosine in urine

Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) has been widely used as a biomarker of oxidative DNA damage. Measurements of 8-OHdG in urinary samples are challenging owing to the low level of 8-OHdG and the complex matrix. In this study, a novel molecularly imprinted polymer (MIP) monolithic column was synthesized with guanosine as a dummy template which was used as the medium for in-tube solid-phase microextraction (SPME). In-tube SPME coupled with HPLC/UV detection for extraction and determination of urinary 8-OHdG was developed. The synthesized MIP monolithic column exhibited high extraction efficiency owing to its greater phase ratio with convective mass transfer and inherent selectivity. The enrichment factor for 8-OHdG was found to be 76 and the limits of detection and quantification of the method for urinary samples were 3.2 nmol/L (signal-to-noise ratio 3) and 11 nmol/L (signal-to-noise ratio 10), respectively. The MIP^’s selectivity also made the sample preparation procedure and chromatographic separation much easier. The linear range of the proposed method was from 0.010 to 5.30 μmol/L (r = 0.9997), with a relative standard deviation of 1.1–6.8%, and the recovery for spiked urine samples was 84 ± 3%. The newly developed method was successfully applied to determine urinary samples of healthy volunteers, coking plant workers, and cancer patients. The 8-OHdG level in cancer patients was significantly higher than that in healthy people.     

Determination of polycyclic aromatic hydrocarbons in sediment by liquid chromatography-atmospheric pressure photoionization-mass spectrometry.

We describe a method for the simultaneous determination of 12 kinds of polycyclic aromatic hydrocarbons (PAHs) in sediment based on liquid chromatography-atmospheric pressure photoionization-mass spectrometry (LC/APPI/MS). The method consists of PAH extractions by ultrasonics, clean-up by a solid-phase extraction procedure and determination by LC/APPI/MS. The limits of the determination for PAHs in sediment using the proposed method ranged from 0.06 to 0.9 mg/kg. PAHs were detected by this method in sediment samples on the mg/kg level.     

自制混合型小柱净化-高效液相色谱-串联质谱法同时测定尿液中有机磷酸酯代谢物和8-羟基-2'-脱氧鸟苷

建立了基于自制混合型小柱的样品净化-高效液相色谱-串联质谱同时测定7种有机磷酸酯（OPEs）主要代谢产物及生物标志物8-羟基-2'-脱氧鸟苷（8-OHdG）的分析方法。样品经乙腈提取后用自制小柱富集净化,以乙腈-0.2%（v/v）氨水溶液作为流动相进行梯度洗脱,在多反应监测模式下进行定性和定量分析。结果显示,8种目标物在0.1~200 μg/L范围内呈良好的线性关系,7种OPEs代谢物的回收率为52.36%~114.56%,8-OHdG回收率为88.63%~97.72%。将该方法应用于人体尿液实际样品中,7种OPEs代谢物和8-OHdG的检出范围分别为6.24~46.07 μg/L和5.90~16.71 μg/L,8-OHdG与7种OPEs代谢物总含量之间存在显著相关性。该方法操作简单、灵敏度高、准确性好、重现性强,可为更全面地评价人体内OPEs暴露水平及机体损伤提供可靠的技术支持。     

超高效液相色谱-串联质谱法测定氧化损伤标志物8-羟基脱氧鸟苷

发展了一种超高效液相色谱-串联质谱法(UPLC-MS/MS)检测脱氧核糖核酸(DNA)分子中8-羟基脱氧鸟苷(8-OHdG)的方法。DNA分子在酶解过程中,脱氧鸟苷(dG)易被氧化形成8-OHdG,从而使得8-OHdG的检测结果不准确。通过加入甲磺酸去铁铵作为保护剂,有效地避免了酶解过程造成的dG氧化。酶解液通过超滤膜(截留相对分子质量为3 000的分子)处理,有效去除大量蛋白分子后,直接进行UPLC-MS/MS测定。采用外标法定量,在17.6～1 400fmol范围内,8-OHdG的峰面积与其物质的量具有良好的线性关系,相关系数为0.999 8。利用本方法测定了小牛胸腺DNA中8-OHdG的含量(用比值8-OHdG/106dG表示)为12.9±2.35,与前人报道的检测结果一致。本方法也可以应用于评价各种氧化因素引起的DNA氧化损伤。     

Evaluation of biological monitoring markers using genomic and proteomic analysis for automobile emission inspectors and waste incinerating workers exposed to polycyclic aromatic hydrocarbons or 2,3,7,8,-tetracholrodedibenzo-p-dioxins

In this study, we investigated the effects of PAHs and dioxin on mRNA and plasma protein expression using genomic and proteomic analysis for automobile emission inspectors and waste incineration workers. About 54 workers from automobile emission inspection offices, 31 workers from waste incinerating company and 84 unexposed healthy subjects were enrolled in this study. Urine and air samples were collected and analyzed by HPLC and GC/MS. Comet assays were carried out to evaluate any DNA damage in mononuclear and polynuclear cells. A significant difference in Olive tail moments in mononuclear cells was observed between exposed and control subjects (P < 0.0001). To examine the differences of the gene expression profile in automobile emission inspectors and waste incineration workers, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 total genes. The gene expression profiles showed that 11 genes were up-regulated and 4 genes were down-regulated in waste incinerating workers as compared with controls. Plasma proteins were analyzed by 2-dimentional electrophoresis with pH 3-10 NL IPG Dry strip. The protein expression profiles showed that 8 proteins were up- regulated and 1 protein, haptoglobin, was down- regulated in automobile emission inspectors and waste incineration workers. Serum paraoxonase/ arylesterase was found only in the plasma of waste incineration workers. The expression of genes and proteins involved in oxidative stress were up-regulated in both automobile emission inspectors and waste incineration workers. Several proteins, such as transthyrethin, sarcolectin and haptoglobin, that were highly up- or down-regulated, could serve as biological monitoring markers for future study.     

Urinary analysis of 8-oxoguanine, 8-oxoguanosine, fapy-guanine and 8-oxo-2'-deoxyguanosine by high-performance liquid chromatography-electrospray tandem mass spectrometry as a measure of oxidative stress

A sensitive and specific assay aimed at measuring the oxidized nucleic acids, 8-oxoguanine (8-oxoGua), fapy-guanine (Fapy-Gua), 8-oxoguanosine (8-oxoGuo), 8-oxo-2'-deoxyguanosine (8-oxodG) has been developed by coupling reversed phase liquid chromatography (HPLC) with electrospray tandem mass spectrometry detection (MS/MS) and isotope dilution. The HPLC-MS/MS approach with multiple reaction monitoring (MRM) allowed for the sensitive determination of 8-oxoGua, Fapy-Gua, 8-oxoGuo, and 8-oxodG in human urine samples. There is no sample preparation needed except for the addition of buffer and (13)C- and (15)N-labeled internal standards to the urine prior to sample injection into the HPLC-MS/MS system. This method was tested in urine samples from non-smokers, smokers, non-smokers with chronic kidney disease (CKD) and smokers with CKD, to assess the level of oxidative damage to nucleic acids. Markers of both RNA and DNA damage were significantly increased in the smokers with and without CKD compared to their respective control subjects. These findings suggest that a highly specific and sensitive analytical method such as isotope dilution HPLC-MS/MS may represent a valuable tool for the measurement of oxidative stress in human subjects.     

Detection of a urinary biomaker for oxidative DNA damage 8-hydroxydeoxyguanosine by capillary electrophoresis with electrochemical detection

8-Hydroxydeoxyguanosine (8-OHdG) is present in urine as a result of oxidative DNA damage associated with age-related diseases such as cancer. In this report a method is presented for the detection of 8-OHdG in human morning urine utilizing capillary electrophoresis with electrochemical detection (CEEC). The limit of detection for a aqueous standard of 8-OHdG is 50 nM (signal to noise ratio S/N = 3). A single solid-phase extraction (SPE) step with a C18 column is used for sample cleanup and 20-fold preconcentration of the urine before analysis by CEEC. Optimized conditions for analysis of extracted urine are E(app) = 0.5 V vs. Ag/AgCl with 20 mM sodium borate/20% MeOH v/v, pH 9, as the background electrolyte, and a separation voltage of 22 kV. The concentration of 8-OHdG varied from 6 to 86 nM with an average value of 42 +/- 26.9 nM for four healthy female and four healthy male subjects between the ages of 23 and 43.     

Determination of urinary S-phenylmercapturic acid, a specific metabolite of benzene, by liquid chromatography/single quadrupole mass spectrometry

A high-performance liquid chromatography/single quadrupole mass spectrometry (LC/MS) method is described for the determination of urinary S-phenylmercapturic acid (S-PMA), a specific metabolite of benzene. Urine samples were spiked with [13C6]S-PMA (used as the internal standard) and acidified; then they were purified by solid-phase extraction (SPE) on C18 cartridges. Analyses were conducted on a reversed-phase column by gradient runs with 1% aqueous acetic acid/methanol mixtures at different proportions as the mobile phase. The detector was used in electrospray negative ion mode (ESI-), the ions m/z 238 for S-PMA and 244 for [13C6]S-PMA being recorded simultaneously. The detection limit (for a signal-to-noise ratio = 3) was 0.2 microg/L, thus allowing for the measurement of background excretion of S-PMA in the general population. The use of the internal standard allowed us to obtain good precision (CV% values < 3%) and a linear calibration curve within the range of interest for monitoring occupational exposure to benzene (up to 500 microg/L). The method was applied to assay the metabolite concentration in a group of 299 workers (68 smokers and 231 non-smokers) occupationally exposed to relatively low levels of benzene (environmental concentration = 0.4-220 microg/m3, mean 11.4 microg/m3 and 236 non-exposed subjects (134 smokers and 102 non-smokers). The results clearly showed that smoking must be taken into account for the correct interpretation of the results of S-PMA measurements for the assessment of work-related benzene exposure. When only non-smokers were selected, the mean excretion of S-PMA was significantly higher in workers exposed to benzene (1.2 +/- 0.9 microg/g creatinine) than in the control group (0.7 +/- 0.6 microg/g creatinine) (p < 0.001), thus confirming the role of S-PMA as a biomarker of benzene on a group basis, even for relatively low exposure degrees.     

Determination of N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) in urine by high-performance liquid chromatography combined with mass spectrometry

A simple, sensitive and specific method for the quantification of N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) in urines of the general population and of exposed workers has been developed. Samples were first diluted with phosphate buffer followed by purification by solid-phase extraction (SPE) with SAX columns prior to analysis by liquid chromatography/mass spectrometry (LC/MS). An external standard was used for the quantification together with selected ion monitoring of m/z 219, [M-H]-. The linear calibration curve showed a good correlation (r2 = 0.98, p < 0.001) between 0.1-110 mg/L of AMCC, and the detection limit was calculated to be 2 microg/L. The within-day and between-day precision, calculated for exposed workers and general population AMCC levels in urine samples, were determined as 1.2 and 3.6%, respectively (as relative standard deviation (RSD)).     

Simultaneous determination of two serine proteinases by hydrophobic interaction chromatography

Summary An LC clean-up procedure based upon a complexation between polycyclic aromatic hydrocarbons (PAHs) and silica with chemically bonded 2,4-dinitroaniline has been combined with GC/MS. The LC pre-separation makes it possible to obtain a relatively clean fraction of PAHs free from alkanes, alkylbenzenes and naphthalenes, PCBs, chlorinated pesticides and many other interfering compounds. This fraction has been analyzed using capillary GC and mass selective detector (MSD). Substantial improvement of the MS spectra of PAHs with three or more fused benzene rings is achieved.     

A method for routine quantitation of urinary 8-hydroxy-2'-deoxyguanosine based on solid-phase extraction and micro-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

8-Hydroxy-2'-deoxyguanosine (8OHdG), one of the major oxidative DNA lesions induced by radical agents, is commonly used as a biomarker for oxidative stress, nowadays preferably in urine. In the absence of a commercially available internal standard a micro-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (micro-HPLC/ESI-MS/MS) method, suitable for routine analysis of 8OHdG in human urine using external calibration, was developed. Evaluation of the matrix effect showed that the method allows highly sensitive and accurate quantitation despite the absence of an internal standard. HPLC analysis was performed using gradient elution at a flow rate of 10 microL min(-1) using a capillary reversed-phase column and an injection volume of 0.5 microL, with detection of 8OHdG in positive multiple reaction monitoring (MRM) mode. The absolute limit of detection was 0.35 fmol using m/z 168 as a quantifier (fragment) ion. A linear (R2> 0.999) calibration curve in urine was obtained over a range 0.2-10 ng mL(-1). This method is about 20 times more sensitive than previously described procedures, and is characterized by high accuracy (mean 90%) and good reproducibility (RSD <10%). The optimized method was applied to determination of 8OHdG in 18 urinary samples derived from three healthy volunteers. 8OHdG urinary excretion ranged from 3.0-7.9 microg/day, and a large intra-individual variation was found. This method, which effectively circumvents the need for isotopically labeled 8OHdG (internal standard), is suitable for routine monitoring of exposure to DNA-damaging factors in a large number of subjects.     

Determination of urinary 8-hydroxy-2′-deoxyguanosine by capillary electrophoresis with molecularly imprinted monolith in-tube solid phase microextraction

<正>Urinary 8-hydroxy-2'-deoxyguanosine(8-OHdG) is an excellent marker of oxidative DNA damage.In this study,employing guanosine as dummy template a novel molecularly imprinted(MIP) monolithic capillary column had been synthesized,and that was used as medium of in-tube solid phase microextraction(SPME).Coupled with capillary electrophoresis-electrochemical detection(CE-ECD),the system of extraction and detection of 8-OHdG in urinary sample had been developed.Because of its greater phase ratio combined with convective mass transfer and inherent selectivity,the MIP monolithic column exhibited high extraction efficiency on target analyte with the limit of detection(LOD) and quantity(LOQ) reached 2.6 nmol/L(S/N = 3) and 8.6 nmol/L(S/N= 10),respectively.The linear range was from 10 nmol/L to 1.5μmol/L(r = 0.9999) with relative standard deviation(RSD) 3.7%for peak current,and 0.5%for migration time,and the average recovery of spiked 20-200 nmol/L 8-OHdG was 85%±3.5%(n = 6).This highly sensitive method was applied to analysis of 8-OHdG in urinary samples from healthy volunteers,coking plant workers and lung cancer patients.     

Determination of 8‐oxo‐2′‐deoxyguanosine and creatinine in murine and human urine by liquid chromatography/tandem mass spectrometry: application to chemoprevention studies

Urinary 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG) represents a non‐invasive biomarker for oxidative stress and may be useful for monitoring chemotherapeutic and chemopreventive interventions associated with cancer‐related alterations in oxidative stress. We describe the development and validation of two separate liquid chromatography/tandem mass spectrometry (LC/MS/MS) selected reaction monitoring (SRM) methods for the determination of 8‐oxodG and creatinine in both murine and human urine using stable isotope labelled internal standards. Levels of 8‐oxodG were normalised to creatinine. The LC/MS/MS methods were applied to two chemoprevention studies utilising tea polyphenols in humans and TRAMP (TRansgenic Adenocarcinoma of the Mouse Prostate) mice. Patients with benign prostatic hyperplasia received 1 g/day of green tea polyphenols (GTP), 1 g/day of black tea theaflavins (BTT) or no treatment for 4 weeks. TRAMP mice received GTP (0.05% in drinking water) for 4 or 25 weeks. Prostate pathology in TRAMP mice was not affected by GTP. Levels of 8‐oxodG were not altered by tea polyphenols in either mice or humans. In TRAMP mice, urinary 8‐oxodG levels were elevated with increasing age (p < 0.0001) but not changed by the presence of prostate tumours. In conclusion, the LC/MS/MS SRM methods described here are ideally suited for the accurate determination of 8‐oxodG and creatinine in urine samples from both clinical and pre‐clinical studies. Copyright © 2008 John Wiley & Sons, Ltd.     

Quantification of urinary N-acetyl-S- (propionamide)cysteine using an on-line clean-up system coupled with liquid chromatography/tandem mass spectrometry

Acrylamide has been reported to be present in high-temperature processed foods and normal processed food intake could lead to significant acrylamide exposure. Acrylamide in vivo can be conjugated with glutathione in the presence of glutathione transferase. This conjugation product is further metabolized and excreted as N-acetyl-S-(propionamide)cysteine (NASPC) in the urine. NASPC could be considered a biomarker for acrylamide exposure. The objective of this study was to develop a highly specific, rapid and sensitive method to quantify urinary NASPC, serving as a biomarker for acrylamide exposure assessment. Isotope-labeled [13C3]NASPC was successfully synthesized and used as an internal standard. This urine mixture was directly analyzed using a newly developed liquid chromatographic/tandem mass spectrometric method coupled with an on-line clean-up system. The detection limit for this method was estimated as < 5 microg l(-1)(0.4 pmol) on-column. The method was applied to measure the urinary level of NASPC in 70 apparently health subjects. The results showed that the NASPC urinary level was highly associated with smoking. Smokers had a significantly higher urinary NASPC level (135 +/- 88 microg g(-1) creatinine) than non-smokers (76 +/- 30 microg g(-1) creatinine). A highly sensitive and selective LC/MS/MS isotope dilution method was successfully established. With an on-line clean-up system, this system is capable of routine high-throughput analysis and accurate quantitation of NASPC in urine. This could be a useful tool for health surveillance for acrylamide exposure in a population for future study.     

Quantitative analysis of amyloid beta peptides in cerebrospinal fluid of Alzheimer's disease patients by immunoaffinity purification and stable isotope dilution liquid chromatography/negative electrospray ionization tandem mass spectrometry

The 40 and 42 amino-acid residue forms of amyloid beta (Abeta(1-40) and Abeta(1-42)) in cerebrospinal fluid (CSF) have been proposed as potential biomarkers of Alzheimer's disease (AD). Quantitative analyses of Abeta peptides in CSF have relied almost exclusively on the use of immunoassay-based assays such as the enzyme-linked immunosorbent assay (ELISA) procedure. However, due to the ability of the Abeta peptides to readily self-aggregate or bind to other proteins and glassware, such analyses are extremely challenging. Analyses are further complicated by the potential of the peptides to undergo post-translational modifications and the possibilities for cross-reaction in the ELISA assays with endogenous components of the CSF. An approach based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) has now been developed which overcomes these methodological issues. The key steps in implementing this new approach involved immunoaffinity purification coupled with the use of [15N]-labeled Abeta peptides as internal standards, a basic LC mobile phase, negative ion electrospray ionization, and a basic solvent for dissolving the peptides and washing the injection needle to prevent carryover of analytes during multiple injections on the LC/MS system. The validated method had limits of quantitation of 44 fmol/mL (200 pg/mL) for Abeta(1-42) and 92 fmol/mL (400 pg/mL) for Abeta(1-40). An excellent correlation was found between the LC/MS/MS assay and an ELISA assay for Abeta(1-42) in human CSF (r2 = 0.915), although less correlation was observed for Abeta(1-40) (r2 = 0.644). Mean CSF Abeta(1-42) concentrations for samples collected 2 weeks apart from a limited number of AD patients provided additional confidence in the reproducibility of the LC/MS/MS assay. Concentrations for duplicate samples from AD patients were slightly higher than most previously reported values (mean 1.06 +/- 0.25 ng/mL; n = 7). Abeta(1-40) concentrations in duplicate samples obtained from AD patients were also reproducible but were found to be slightly lower than most previously reported values (mean 6.36 +/- 3.07 ng/mL; n = 7). Consistent with literature reports, mean Abeta(1-42) concentrations were found to be lower in AD patients compared with the normal subjects (mean 1.49 +/- 0.59 ng/mL; n = 7), whereas there was no difference in Abeta(1-40) concentrations between AD patients and normal subjects (mean 5.88 +/- 3.03 ng/mL; n = 7). The accuracy and precision of the LC/MS assay mean that it will be a useful complement to existing ELISA assays for monitoring therapeutic interventions designed to modulate CSF Abeta(1-42) concentrations in individual AD patients. Moreover, the introduction of stable isotope labeled internal standards offers the potential to achieve a more rigorous account of the influence of methodological effects related to sample collection and processing.