Single-step Solid-phase Extraction Research Articles

A novel single step solid-phase extraction combined with bromine derivatization method for rapid determination of acrylamide in coffee and its products by stable isotope dilution ultra-performance liquid chromatography tandem triple quadrupole electrospray ionization mass spectrometry

This work aimed to establish a novel determination method for acrylamide in coffee and its products by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Acrylamide in samples were prepared by a single-step solid-phase extraction clean-up using mixed mode sorbents. The bromine derivatization efficiency of acrylamide and its internal standard were improved at an acidic condition. After derivation, the retention capability of acrylamide and its resistance to interference were significantly improved. The limit of detection (LOD) and the limit of quantification (LOQ) were 1.2 and 4 μg/kg for roasted and instant coffees, while they were 0.24 and 0.8 μg/kg for ready-to-drink coffees. The average recoveries for acrylamide ranged from 99.3 to 102.2% in coffee and its products. All the results showed that the developed method was simple, quick, specific and suitable for screening and determination of acrylamide in batch samples of coffee and its products.

Targeted Metabolomics: The LC-MS/MS Based Quantification of the Metabolites Involved in the Methylation Biochemical Pathways.

Biochemical methylation reactions mediate the transfer of the methyl group regulating vital biochemical reactions implicated in various diseases as well as the methylation of DNA regulating the replication processes occurring in living organisms. As a finite number of methyl carriers are involved in the methyl transfer, their quantification could aid towards the assessment of an organism’s methylation potential. An Hydrophilic Interaction Chromatography-Liquid Chromatography Multiple Reaction Monitoring (HILIC-LC-MRM) mass spectrometry (MS) methodology was developed and validated according to Food & Drug Administration (FDA), European Medicines Agency (EMA), and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) for the simultaneous determination of nine metabolites i.e., B12, folic acid, 5-methyltetrahydrofolate, S-adenosylmethionine, S-adenosylhomocysteine, betaine, phosphocholine, N,N-dimethylglycine, and deoxythymidine monophosphate in human blood plasma. The sample pretreatment was based on a single step Solid-phase extraction (SPE) methodology using C18 cartridges. The methodology was found to accurately quantitate the analytes under investigation according to the corresponding dynamic range proposed in the literature for each analyte. The applicability of the method was assessed using blood donor samples and its applicability demonstrated by the assessment of their basal levels, which were shown to agree with the established basal levels. The methodology can be used for diagnostic purposes as well as for epigenetic screening.

Differences in xylovolatiles composition between chips or barrel aged wines

Aim: In this research xylovolatile aromatic compounds were determined to highlight any compositional differences between wines aged in barrels and those produced using oak chips.Methods and results: Approximately 200 wines aged using oak chips or wood barrels were analysed, among which about 50 were aged at the experimental winery of the Research Centre of Viticulture and Enology in Asti (Piedmont, Italy). 17 different types of commercial oak chips were used in order to obtain a subset of reference samples. Several factors were considered, including degree of oak wood toasting, wood geographical origin and the type of wine (both red and white). Sample preparation for GC-MS analysis was performed by single-step solid phase extraction, using polymeric SPE cartridges. More than 60 volatile molecules from oak were identified. Analytical results were explained using multivariate statistical analysis. A preliminarily step was carried out using principal component analysis (PCA), which showed interesting compositional differences between barrel and chip-aged wines with regards to methylvanillate, ethylvanillate, as well as furan derivative compounds. Further statistical analysis highlighted the clear impact of the type of wine (red or white) on the extraction of xylovolatile compounds from oak chips. Subsequently, in order to test if and how the selected chemical explanatory variables allow wine treatments to be discriminated, and to predict which group a new observation will belong to, a discriminant analysis (DA) was carried out on an independent dataset. More than 96 % of samples were correctly classified.Conclusions: Significant differences were highlighted between barrel- and chip-aged wines with regards to xylovolatile compounds. The analysis showed that several factors may influence the amount of aromatic compounds extracted from chips and barrels, especially the matrix composition. It was possible to deduce the aging process that the wines had undergone by selecting key molecules using multivariate methods.Significance and impact of the study: Via a suitable GC-MS method and a chemometric approach for the identification of discriminant xylovolatiles in wood aged wines, this study offers promising perspectives and useful tools for routine fraud inspection aiming at classifying wines according to their aging process.

A SPE-HPLC-MS/MS method for the simultaneous determination of prioritised pharmaceuticals and EDCs with high environmental risk potential in freshwater

This work describes the development, optimisation and validation of an analytical method for the rapid determination of 17 priority pharmaceutical compounds and endocrine disrupting chemicals (EDCs). Rather than studying compounds from the same therapeutic class, the analyses aimed to determine target compounds with the highest risk potential (with particular regard to Scotland), providing a tool for further monitoring in different water matrices. Prioritisation was based on a systematic environmental risk assessment approach, using consumption data; wastewater treatment removal efficiency; environmental occurrence; toxicological effects; and pre-existing regulatory indicators. This process highlighted 17 compounds across various therapeutic classes, which were then quantified, at environmentally relevant concentrations, by a single analytical methodology. Analytical determination was achieved using a single-step solid phase extraction (SPE) procedure followed by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The fully optimised method performed well for the majority of target compounds, with recoveries >71% for 15 of 17 analytes. The limits of quantification for most target analytes (14 of 17) ranged from 0.07 ng/L to 1.88 ng/L in river waters. The utility of this method was then demonstrated using real water samples associated with a rural hospital/setting. Eight compounds were targeted and detected, with the highest levels found for the analgesic, paracetamol (at up to 105,910 ng/L in the hospital discharge). This method offers a robust tool to monitor high priority pharmaceutical and EDC levels in various aqueous sample matrices.

METHOD DEVELOPMENT AND VALIDATION OF SELECTIVE AND HIGHLY SENSITIVE METHOD FOR DETERMINATION OF APIXABAN IN HUMAN PLASMA USING LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

Objective: The present research work aims to develop and validate a selective and highly sensitive method for the determination of apixaban in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
 Methods: 200 µl of sodium heparin plasma samples were acidified and clean-up was performed by using solid-phase extraction (SPE). Apixaban 13C D3 was used as an internal standard (deuterated) to lower the relative matrix effects and a single step SPE was employed for sample clean up. 10 µl of SPE eluent was loaded onto Hypersil Beta Basic C18, 100×4.6 mm, 5 µ column for highly selective chromatographic separation using an isocratic mobile phase. 2 mmol ammonium acetate in water and acetonitrile were delivered by using a quaternary low-pressure gradient pump without premixing at a minimum flow rate of 0.50 ml/min.
 Results: LC-MS/MS method was successfully developed and validated to demonstrate the lowest detection limit of 0.05 ng/ml and a linear dynamic range from 1-250 ng/ml with r2>0.99. Method development and validation results proved that the method is selective and highly sensitive for the determination of apixaban in human plasma using LC-MS/MS.
 Conclusion: Current method can be applied for both therapeutic drug monitoring (TDM) and pharmacokinetic (PK) study analysis.

Development and comparison of single-step solid phase extraction and QuEChERS clean-up for the analysis of 7 mycotoxins in fruits and vegetables during storage by UHPLC-MS/MS

A robust and sensitive UHPLC-MS/MS method was developed for the analysis of seven mycotoxins in fruits and vegetables. The variations of seven mycotoxins concentrations during storage were also determined for the first time. Solid phase extraction (SPE) and QuEChERS clean-up were compared, and extraction was finally performed with acetonitrile under acidic conditions followed by HLB SPE purification. Seven mycotoxins were separated on a C18 column by gradient elution, scanned by ESI+ and ESI− dynamic switching and detected with MRM mode. LOD and LOQ, matrix effect, accuracy and precision of the developed method were investigated. Results were linear in their concentration ranges for all mycotoxins with R2 being greater than 0.999. LODs and LOQs were ranged from 0.05 to 3.0 μg/kg and 0.2 to 10.0 μg/kg, respectively. Average recoveries were between 81.1% and 116% with intraday- and interday-precision being in the ranges of 3.0–6.2% and 4.2–6.1%, respectively. The validated method provides sufficient selectivity, sensitivity and accuracy to screen for seven mycotoxins at trace levels, without using standard addition or matrix-matched calibration to compensate for matrix effects. The method was successfully applied for mycotoxins analysis in fruits and vegetables. None were detected in fresh samples, but AOH and AME were detected in strawberry, and TeA was detected in tomato fruits during long-term storage. Their concentrations were in the range of 3.6–165.3 μg/kg from 3 days to 60 days and significantly increased along with storage time.

Determination of Squalene in Organic Extra Virgin Olive Oils (EVOOs) by UPLC/PDA Using a Single-Step SPE Sample Preparation

In this paper, a simple extraction and fast detection procedure was used to determine squalene (SQ) in extra virgin olive oils (EVOOs). SQ was purified from EVOOs by an efficient single-step solid phase extraction (SPE), and its content was determined using an UPLC-PDA instrument. The adopted technique was evaluated for accuracy, linearity, sensitivity, and repeatability. The precision of the SPE extraction was satisfactory and the mean recoveries were 91.9 ± 0.4 and 96.3 ± 0.3 % for 25 and 50 mg L−1 level of addition, respectively. The selected chromatographic conditions allowed a very fast SQ determination; in fact, it was well separated in ∼0.54 min with good resolution. The UPLC method showed a good linearity in the range 50–500 mg L−1 (R 2 = 0.9998). Method sensitivity was evaluated by measuring the limits of detection (LOD) that was 0.3 mg L−1. The method was utilized for SQ determination in 33 different organic EVOO samples, coming from different countries (Turkey, Tunisia, Spain, Portugal, Greece, USA, Slovenia, Albania, Israel, Italy) and the data were statistically evaluated.

A single-step solid phase extraction for the simultaneous determination of 8 mycotoxins in fruits by ultra-high performance liquid chromatography tandem mass spectrometry

A simple and rapid extraction procedure for the simultaneous determination of eight mycotoxins (Alternaria toxins, ochratoxin A, patulin, citrinin) in a variety of fruit matrices has been developed using ultra high performance liquid chromatography coupled to tandem mass spectrometry. The procedure involves a one-step cleanup using homemade solid phase extraction (SPE) cartridges. By comparative evaluation among six various adsorbents (C18, PSA, HLB, MCX, Silica, NH2), the combination of MCX and NH2 was found to provide the most effective cleanup, removing the greatest number of matrix interferences and also allowing the quantification of all analyzed mycotoxins in fruits. The optimized extraction conditions including acidified aqueous acetonitrile and an additional salt-out step using NaCl were employed before SPE cleanup. Method validation was performed by analyzing samples spiked at three levels (LOQ, 2 LOQ and 10 LOQ). Four fruits including apple, sweet cherry, tomato and orange fruits were selected, and accuracy (recovery%), precision (RSD%), limits of quantification (LOQ), linearity and matrix effect were evaluated during validation. Matrix-matched linearity with correlation coefficients ≥0.9921 was established in the range of 5–200ngmL−1 for patulin and 1–200ngmL−1 for other mycotoxins, respectively. Recoveries between 74.2% and 102.4% and relative standard deviations lower than 4.7% were obtained for all tested fruits. The matrix effect observed was low (≤±17%) in all three fruit matrixes with the exception of orange, for which strong ion suppression was observed for alternariol (25.3%), ochratoxin A (31.6%) and citrinin (40.3%). Therefore, matrix-matched calibration was used for a correct quantification in order to compensate for matrix effect. The limits of quantification (LOQ), ranging from 1 to 5μgkg−1 depending on mycotoxins type, were always lower than maximum permitted levels for every regulated mycotoxin by the current European legislation.

Solid-phase extraction using bis(indolyl)methane-modified silica reinforced with multiwalled carbon nanotubes for the simultaneous determination of flavonoids and aromatic organic acid preservatives.

A novel bis(indolyl)methane-modified silica reinforced with multiwalled carbon nanotubes sorbent for solid-phase extraction was designed and synthesized by chemical immobilization of nitro-substituted 3,3'-bis(indolyl)methane on silica modified with multiwalled carbon nanotubes. Coupled with high-performance liquid chromatography analysis, the extraction properties of the sorbent were evaluated for flavonoids and aromatic organic acid compounds. Under optimum conditions, the sorbent can simultaneously extract five flavonoids and two aromatic organic acid preservatives in aqueous solutions in a single-step solid-phase extraction procedure. Wide linear ranges were obtained with correlation coefficients (R(2) ) ranging from 0.9843 to 0.9976, and the limits of detection were in the range of 0.5-5 μg/L for the compounds tested. Compared with the silica modified with multiwalled carbon nanotubes sorbent and the nitro-substituted 3,3'-bis(indolyl)methane-modified silica sorbent, the developed sorbent exhibited higher extraction efficiency toward the selected analytes. The synergistic effect of nitro-substituted 3,3'-bis(indolyl)methane and multiwalled carbon nanotubes not only improved the surface-to-volume ratio but also enhanced multiple intermolecular interactions, such as hydrogen bonds, π-π, and hydrophobic interactions, between the new sorbent and the selected analytes. The as-established solid-phase extraction with high-performance liquid chromatography and diode array detection method was successfully applied to the simultaneous determination of flavonoids and aromatic organic acid preservatives in grape juices with recoveries ranging from 83.9 to 112% for all the selected analytes.

A novel mixed-mode solid phase extraction coupled with LC–MS/MS for the re-evaluation of free 3-nitrotyrosine in human plasma as an oxidative stress biomarker

3-Nitrotyrosine (3-NT) has been widely adopted as a biomarker of oxidative stress. However, an enormous range of reported concentrations of 3-NT in biological matrices indicated an underlying methodological problem. Consequently, our understanding of tyrosine nitration in vivo and its significance as a biomarker may have been confounded. Here we report a fast, specific and sensitive LC–MS/MS method to accurately quantify free 3-NT in human plasma. For the first time, a single-step solid phase extraction of 3-NT using mixed-mode sorbent in a 96-well plate was developed after significant optimization. Complete chromatographic separation of 3-NT from other tyrosine analogs was achieved on a PFPP column (150mm, 2.1mm, 3μm) with a cycle time of 10min. The developed method was validated in terms of linearity, sensitivity, specificity, precision, accuracy, matrix effect, carryover, analyte stability and reference interval. The lower limit of quantification of the method was 5pg/ml for plasma 3-NT, which represents a significant sensitivity improvement over reported methods. No artifactual 3-NT formation was observed, and the assay was not affected by 40 likely interferences. The average intra- and inter-assay variances were 3.4% and 3.7%, respectively. Of note, the reference interval for a healthy population was established to be 2.0–40.1pg/ml (8.8–177pM) with a mean of 11.1pg/ml (49pM). The mean value is at least 13-fold lower than previously reported mean values. Furthermore, the method was applied to the investigation of biological variation (n=15) over a three week period and no statistical differences were found. The solid phase extraction in a 96-well format and a fast LC–MS/MS delivered a practical, precise and accurate tool well-suited to quantify free plasma 3-NT in a large number of biological samples.

Simultaneous determination of PPCPs, EDCs, and artificial sweeteners in environmental water samples using a single-step SPE coupled with HPLC–MS/MS and isotope dilution

A high-throughput method for the simultaneous determination of 24 pharmaceuticals and personal care products (PPCPs), endocrine disrupting chemicals (EDCs) and artificial sweeteners (ASs) was developed. The method was based on a single-step solid phase extraction (SPE) coupled with high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) and isotope dilution. In this study, a single-step SPE procedure was optimized for simultaneous extraction of all target analytes. Good recoveries (≥70%) were observed for all target analytes when extraction was performed using Chromabond® HR-X (500mg, 6mL) cartridges under acidic condition (pH 2). HPLC–MS/MS parameters were optimized for the simultaneous analysis of 24 PPCPs, EDCs and ASs in a single injection. Quantification was performed by using 13 isotopically labeled internal standards (ILIS), which allows correcting efficiently the loss of the analytes during SPE procedure, matrix effects during HPLC–MS/MS and fluctuation in MS/MS signal intensity due to instrument. Method quantification limit (MQL) for most of the target analytes was below 10ng/L in all water samples. The method was successfully applied for the simultaneous determination of PPCPs, EDCs and ASs in raw wastewater, surface water and groundwater samples collected in a local catchment area in Singapore. In conclusion, the developed method provided a valuable tool for investigating the occurrence, behavior, transport, and the fate of PPCPs, EDCs and ASs in the aquatic environment.

Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of glucocorticoid residues in edible tissues of swine, cattle, sheep, and chicken

A confirmatory and quantitative method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the presence of eight glucocorticoids (prednisone, prednisolone, hydrocortisone, methylprednisolone, dexamethasone, betamethasone, beclomethasone, and fludrocortisone) in the muscles and livers of swine, cattle, and sheep and the muscle of chicken is described. After deconjugation in alkali media, samples were extracted with ethyl acetate for glucocorticoids followed by solid-phase extraction clean-up and reconstitution in the LC mobile phase. The hydrolysis procedure with sodium hydroxide was used to reduce handling time. A single-step solid-phase extraction method was optimized which is suitable for the clean-up of the compounds of interest in many diverse tissue matrices. LC separations were performed on a C18 column with gradient elution using acetonitrile and water (containing 0.2% formic acid) and the two epimers betamethasone and dexamethasone were successfully separated. LC-electrospray ionization (ESI)-MS/MS in negative mode with selected reaction monitoring (SRM) mode was performed to improve method sensitivity and reduce matrix interference. Two SRM transitions were used for each compound. The recovery of glucocorticoids spiked at levels of 0.5–16 µg kg−1 ranged from 55% to 107%; the between-day relative standard deviations were no more than 15%. The limits of quantification were 0.5–2.0 µg kg−1 in muscle and 1–4 µg kg−1 in liver. The optimized procedure was successfully applied to monitor the food at the 2008 Summer Olympics Games in Beijing, China, demonstrating the method to be simple, fast, robust, and suitable for identification and quantification of glucocorticoids residues in foods of animal origin.

Detection of extracellular 8-oxo-7,8-dihydro-2′-deoxyguanosine as a biomarker of oxidative damage in X-irradiated fibroblast cultures: optimization of analytical procedure

We have developed a simple methodology, based on single-step solid-phase extraction followed by isocratic high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD), to determine extracellular 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in culture supernatants of normal human dermal fibroblasts. A standard addition method, using externally added 8-oxodG (0.5 and 1 pmol) was employed to eliminate matrix effects arising from the chemically complex, protein-rich medium. Secondly, applying this procedure to X-ray irradiated fibroblasts, we report a significant twofold increase in the levels of 8-oxodG at the radiobiologically relevant dose of 6 Gy. This suggests that extracellular 8-oxodG might be a useful biomarker for oxidative stress following moderate doses of X-irradiation.

Quantification of F2-isoprostane isomers in cultured human lung epithelial cells after silica oxide and metal oxide nanoparticle treatment by liquid chromatography/tandem mass spectrometry

F(2)-isoprostanes are lipid peroxidation products of arachidonic acid in cell membrane and are reliable biomarkers for oxidative stress and cell membrane damage. Nanomaterials are widely used as raw materials in many industries and will have high potentials to be used in life science and medical fields. However, the human health impact of nanoparticles has caused people's great concern. Unfortunately, the mechanisms of cytotoxicity of many nanoparticles are not well defined. By measuring the levels of F(2)-isoprostane isomers in cultured cells after nanoparticle exposure, the information can be used to explain whether the cytotoxicity of nanoparticles is caused by lipid peroxidation and to investigate the biological pathways. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to separate and quantify F(2)-isoprostane isomers in nanoparticle-treated human lung cancer cells. Silica oxide (15nm) and other four metal oxide nanoparticles including Fe(2)O(3) (30nm), Al(2)O(3) (13nm), TiO(2) (40nm) and ZnO (70nm) are chosen in this study. The isotope forms of F(2)-isoprostane isomers, 8-iso-PGF(2alpha)-d(4) and PGF(2alpha)-d(4), were used as internal standard (IS). After human lung epithelial cells were exposed to different nanoparticles for 24h, F(2)-isoprostanes were extracted by a single step solid phase extraction with Oasis HLB cartridge. For the first time, six F(2)-isoprostane isomers were tentatively identified and quantified in human lung epithelial cells. The levels of F(2)-isoprostane isomers in the cells increased after the treatment with nanoparticles. For SiO(2), Fe(2)O(3), and ZnO nanoparticles, F(2)-isoprostane isomers increasing are consistent with nanoparticles' cytotoxicity data. For Al(2)O(3) and TiO(2) nanoparticles, F(2)-isoprostane isomers levels increased even before nanoparticles showed significant cytotoxicity at 100microg/mL concentration in 24h. Based on our best knowledge, this is the first study on the F(2)-isoprostane isomers corresponding to nanoparticles' exposure in vitro. Our study demonstrates that SiO(2) (15nm) nanoparticle showed the highest degree of lipid peroxidation and cell membrane damage among the studied nanoparticles.

Study of urinary 8-hydroxydeoxyguanosine as a biomarker of oxidative DNA damage in diabetic nephropathy patients

Increased oxidative stress induced by hyperglycemia may contribute to the pathogenesis of diabetic complications. Urinary 8-hydroxydeoxyguanosine (8-OHdG) has been reported to serve as a sensitive biomarker of oxidative DNA damage and also of oxidative stress. This article studied oxidative DNA damage in patients with diabetic nephropathy and in healthy control subjects by urinary 8-OHdG evaluations. Contents of 8-OHdG in urine were analyzed by capillary electrophoresis with end-column amperometric detection (CE-AD) after a single-step solid-phase extraction (SPE). Levels of urinary 8-OHdG in diabetic nephropathy patients with macroalbuminuria was significant higher than in control subjects (5.72±6.89 μmol/mol creatinine versus 2.33±2.83 μmol/mol creatinine, P = 0.018). A significant difference of 24 h urinary 8-OHdG excretions exists between the patients with macroalbuminuria and the patients with normoalbuminuria (19.2±16.8 μg/24 h versus 8.1±1.7 μg/24 h, P = 0.015). There was a positive correlation between urinary excretion of 8-OHdG and glycosylated hemoglobin (HbA 1c) ( r = 0.287, P = 0.022). A weak correlation exists between the levels of 8-OHdG and triglyceride ( r = 0.230, P = 0.074). However, the urinary 8-OHdG contents are not correlated with blood pressure and total cholesterol. The increased excretion of urinary 8-OHdG is seen as indicating an increased systemic level of oxidative DNA damage in diabetic nephropathy patients.

Determination of urinary 8-hydroxy-2′-deoxyguanosine in obese patients by HPLC with electrochemical detection

A simple amperometric detector with a platinum tubular electrode was tested for the determination of urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) by high-performance liquid chromatography. Analyte was extracted from human urine using a single-step solid-phase extraction with the average recovery of 71±7%. Analysis was carried out on a C-18 analytical column under isocratic conditions. The limit of detection of 8-OHdG of 3 nM (S/N=3) was comparable with that achieved using the conventional amperometric detector. Procedure was used for the determination of 8-OHdG levels in 10 obese patients (body-mass index>30 kg m −2) yielding the mean concentration 32±27.1 nM of 8-OHdG or 1.85±0.72 μmol 8-OHdG mol −1 creatinine (4.63±1.8 μg 8-OHdG g −1 creatinine), which is not much different from the levels found in healthy individuals.

Determination of urinary oxidative DNA damage marker 8-hydroxy-2′-deoxyguanosine and the association with cigarette smoking

8-hydroxy-2′-deoxyguanosine (8OHdG) has been widely used as a biomarker of oxidative DNA damage in both animal models and human studies. To evaluate the effect of cigarette smoking on oxidative stress, we studied the levels of urinary 8OHdG from smokers and non-smokers and investigated the association with cigarette smoking. The urinary 8OHdG concentrations were determinated by capillary electrophoresis with end-column amprometric detection (CE-AD) after a single-step solid phase extraction (SPE), and then quantitatively expressed as a function of creatinine excretion. To increase the concentration sensitivity, a dynamic pH junction was used and the focusing effect was obvious when using 30mM phosphate (pH 6.50) as sample matrix. The limit of detection is 4.3nM (signal-to-noise ratio S/N=3). The relative standard deviation (R.S.D.) was 1.1% for peak current, and 2.3% for migration time. Based on the selected CE-AD method, it was found that the mean value of urinary 8OHdG levels in the smokers significantly higher than that in non-smokers (31.4±18.9nM versus 14.4±7.6nM, P=0.0004; 23.5±21.3μgg−1 creatinine versus 12.6±13.2μgg−1 creatinine, P=0.028).

Capillary electrophoresis with end-column amperometric detection of urinary 8-hydroxy-2'-deoxyguanosine.

Urinary 8-hydroxy-2'-deoxyguanosine (8OHdG) is an excellent marker of oxidative DNA damage. Until now, urinary 8OHdG has been measured by high-performance liquid chromatography with electrochemical detection. A simple and sensitive method for the analysis of urinary 8OHdG by capillary electrophoresis with end-column amperometric detection has been developed in our laboratory. A single-step solid-phase extraction procedure was optimized and used for extracting 8OHdG from human urine. To improve the sensitivity of this method, a new focusing technique based on a dynamic pH junction was used. The limit of detection was 20 nM (signal-to-noise ratio S/N = 3), the linear range was 50 nM-10 microM, and the correlation coefficient was better than 0.999. The relative standard deviation (RSD) was found to be 0.57% for migration time, and 4.79% for peak current. To show the usefulness of the method, the urinary concentration of 8OHdG in nine healthy persons and ten cancer patients was determined. The urinary concentration of 8OHdG in cancer patients was significantly higher than that in healthy persons.

ANALYSIS OF URINARY 8-HYDROXYDEOXYGUANOSINE BY CAPILLARY ELECTROPHORESIS AND SOLID-PHASE EXTRACTION

Urinary 8-hydroxydeoxyguanosine (8OHdG) has been considered as an excellent marker of individuals at high risk of developing cancer. Until now, urinary 8OHdG has largely been measured by high-performance liquid chromatography with electrochemical detection. A new method for the analysis of urinary 8OHdG by high-performance capillary electrophoresis has been developed and optimized in our laboratory. A single step solid-phase extraction procedure was optimized and used for extracting 8OHdG from human urine. Separations were performed in an uncoated silica capillary (50 cm × 50 μm i.d.) using a P/ACE MDQ system with UV detection. The separation of 8OHdG from interfering urinary matrix components is optimized with regard to pH, applied voltage, pressure injection time and concentration of SDS in running buffer. The detection limit of this method is 0.4 μg/ml, the linear range is 0.8–500 μg/ml, the correlation coefficients levels is better than 0.999. The developed method is simple, fast and good reproducibility, furthermore, it requires a very small injection volumes and low costs of analysis, which makes it possible to provide a new noninvasive assay for an indirect measurement of oxidative DNA damage.

Quantitative determination of an extremely polar compound allantoin in human urine by LC-MS/MS based on the separation on a polymeric amino column

Quantitative determination of allantoin in biological matrix poses a challenge to bioanalysis due to the extreme polarity of allantoin. The molecule exhibits virtually no retention on any of the available hydrophobic HPLC packing materials. In this study, an assay was developed for the LC-MS/MS quantitation of allantoin in human urine using a polymeric amino column to achieve the necessary separation. The urine samples were prepared by a single-step solid phase extraction (SPE) procedure. The extracted samples were analyzed on a jordi-gel DVB polyamine column (operated under an acetonitrile/water gradient with water as the stronger mobile phase) interfaced with a Finnigan TSQ 7000 mass spectrometer. Negative electrospray ionization (ESI) was employed as the ionization source. Allantoin and its internal standard ( 13C 1, 15N 1-allantoin) were detected by use of single reaction monitoring (SRM) mode. The method was validated in the concentration range of 14.6–213 μg ml −1, with within and between run accuracy and precision both <7%. The method has been successfully applied to clinical sample analysis.