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Modifications of genomic DNA may change gene expression and cause adverse health effects. Here we for the first time demonstrate a particle counting immunoassay for rapid and sensitive detection of DNA modifications using benzo[a]pyrenediol epoxide (BPDE)--DNA adducts as an example. The BPDE-adducted DNA is specifically captured by immunomagnetic particles and then isolated from unmodified DNA by applying an external magnetic field. By taking advantage of the fluorescence signal amplification through multiple labeling of captured DNA by OliGreen dye, the captured BPDE--DNA adducts can be quantified by particle counting from fluorescence imaging. This clearly demonstrates that the number of fluorescently countable particles is proportional to the modification content in genomic DNA. It is interesting to note that the background fluorescence signal caused by nonspecific adsorption of OliGreen dye can be more effectively quenched than that induced by the binding of OliGreen dye to ssDNA, allowing for significant reduction in the background fluorescence and further enhancing the detection sensitivity. The developed method can detect trace BPDE--DNA adducts as low as 180 fM in the presence of I billion times more normal nucleotides in genomic DNA and has a dynamic range over 4 orders of magnitude. By using anti-5-methylcytosine antibody, the method is extended to the detection of global DNA methytation. With high sensitivity and specificity, this rapid and easy-to-perform analytical method for DNA modifications shows a broad spectrum of potential applications in genotexical and epigenetic analysis. 10.1021/ac102416f

Monitoring of over-expressed nucleolin in the cytoplasm facilitates early cancer diagnosis. Herein, we present a novel biosensing nanoscaffold based on anti-nucleolin aptamers and polymer-grafted graphene oxides for the fluorescent imaging of nucleolin in the cell cytoplasm, which can distinguish cancer cells from normal cells. [ABSTRACT FROM AUTHOR]

Graphene oxide (GO) has been extensively explored as a promising nanomaterial for applications in biology because of its unique properties. Therefore, systematic investigation of GO toxicity is essential to determine its fate in the environment and potential adverse effects. In this study, acute toxicity, oxidative stress and immunotoxicity of GO were investigated in zebrafish. No obvious acute toxicity was observed when zebrafish were exposed to 1, 5, 10 or 50 mg/L GO for 14 days. However, a number of cellular alterations were detected by histological analysis of the liver and intestine, including vacuolation, loose arrangement of cells, histolysis and disintegration of cell boundaries. As evidence for oxidative stress, malondialdehyde levels and superoxide dismutase and catalase activities were increased and glutathione content was decreased in the liver after treatment with GO. GO treatment induced an immune response in zebrafish, as demonstrated by increased expression of tumor necrosis factor α, interleukin-1 β, and interleukin-6 in the spleen. Our findings demonstrated that GO administration in an aquatic system can cause oxidative stress and immune toxicity in adult zebrafish. To our knowledge, this is the first report of immune toxicity of GO in zebrafish. [ABSTRACT FROM AUTHOR]

Exogenous and endogenous genotoxic carcinogens and their in vivo metabolites may result in DNA damage and cause adverse health effects. It is very difficult to specifically detect trace DNA damage in the presence of a large excess of unmodified nucleosides. Here we report a molecularly imprinted polymer (MIP) nanocomposite, namely nanoMIP, which can be used as a “plastic antibody” for specific recognition of benzo[a]pyrene diol epoxide (BPDE)–DNA adduct. Enhanced binding affinity (880 nM) of nanoMIPs was achieved by using two tailor-made functional monomers. The property of binding kinetics was greatly improved in virtue of the well-defined nanostructure, which was fabricated by initiators for continuous activator regeneration-atom transfer radical polymerization (ICAR-ATRP). The BPDE-adducted DNA can be specifically captured by synthetic nanoMIP. By taking advantage of this antibody-mimicking behavior, we further developed a fluorescently imaged particle counting immunoassay (FIPCIA) method for ultrasensitive detection of BPDE–ssDNA adducts using a laser scanning confocal microscope (LSCM). The number of countable fluorescent dots is proportional to the content of BPDE–ssDNA adducts in the DNA sample. The proposed plastic antibody-based FIPCIA method can detect traces of BPDE–DNA adducts as low as 18 pM in human lung carcinoma A549 cells. This highly-sensitive detection of DNA lesions offers a promising alternative to immunogenic antibody-based immunoassays for genomics and DNA modification analysis. [ABSTRACT FROM AUTHOR]

Abstract: A high-performance liquid chromatography (HPLC) method with evaporative light scattering detection (ELSD) was applied for simultaneous determination of stachyose, sucrose and raffinose in Chinese artichoke (Stachys Sieboldii Miq.). Carbohydrates were separated on a Bondapak NH2 column using a ternary solvent mixture of methanol–acetonitrile–water (55:25:20, v/v/v) as mobile phase. Two different crafts (method 1 and method 2) for extraction and purification of stachyose in Chinese artichoke tubers were detailed and evaluated. Method 1 can meet the purpose of quantification and method 2 is appropriate for the purpose of purification. The content of stachyose in dry tubers was determined to be 236.0mg/g (from method 1) and the purity of the extracted stachyose flour was calculated to be 87.34% (from method 2), respectively. The analytical method fulfills all the standard requirements of linearity, accuracy and precision. Therefore, it is suitable for purification and routine quantification of stachyose in Chinese artichoke. [Copyright &y& Elsevier]

Molecularly imprinted nanotubes for enantioselective drug delivery and controlled release are fabricated by the combination of template synthesis and ATRP grafting. The release of R-propranolol from the imprinted nanotubes in rats is restricted while the release of pharmacologically active S-enantiomer is greatly promoted. [ABSTRACT FROM AUTHOR]

Long-term exposures to environmental toxicants are causative factors for human health effects. DNA damages are of great interest in that they can serve as the potential biomarkers of environmental exposure and mutations. Qualitative and quantitative detection of their level in genomic DNA, are helpful for the understanding of the generation, transformation, repair and fate of DNA damages and for elucidating the mechanism underlying carcinogenesis. Methodologies based on liquid chromatography-mass spectrometry (LC-MS) have shown unique advantages in accurate and highly sensitive analysis of these structurally modified DNA damages. The present review summarizes our current knowledge on LC-MS analysis of genetic DNA damages, with special emphasis on structurally chemical DNA modifications induced by environmental exposure. Advances in LC-MS analysis and monitoring of oxidative DNA lesion, DNA adducts induced by heavy metals, persistent organic pollutants, bisphenols, fine particulate matters, aldehydes and halobenzoquinones are comprehensively summarized. • Highly sensitive LC-MS methods for DNA damages analysis. • Oxidative DNA damages, DNA adducts induced by environmental exposure. • DNA modifications caused by new emerging contaminants including PM2.5 and BPA. • Advances in DNA damage monitoring and adductomics benefited from LC-MS/MS. [ABSTRACT FROM AUTHOR]

Abstract Four DNA bases are essential biochemical elements for coding heritable life blueprint. Meanwhile, organisms utilize modifying enzymes to post chemical groups on DNA bases to regulate gene expression, chromosome structures, and transposase activity, and to defense pathogens. The researches on the biochemical modifications of DNA bases are at the cutting edge of life sciences and chemical biology. Up to date, along with fantastic progresses, liquid chromatography hyphenated with mass spectrometry (LC-MS)-based technologies become popular and powerful tools for chemical structure-dependent identification and quantification of diverse DNA modifications. Here we provided comprehensive review on LC-MS-based technologies and related pretreatment technologies for profiling of epigenetic DNA modifications, mainly focusing on innovative technologies developed for desalting, DNA digestion, and ionization for mass spectrometry detection. We also summarized the epigenetic progresses, which benefited from the development of the LC-MS methodologies and technologies, on the studies of specific DNA modifications, in particular, 5-hydroxymethylcytosine and 5-formylcytosine. Highlights • LC-MS/MS methods for detection of epigenetic DNA modifications. • DNA hydrolysis for generation of LC-MS detectable mononucleosides. • Chemical approaches for enhancing LC-MS signal. • Progresses on epigenetic DNA modifications benefited from LC-MS/MS analysis. [ABSTRACT FROM AUTHOR]

8-Hydroxydeoxyguanosine (8-OHdG) is a widely-used biomarker of oxidative DNA damages. 8-OHdG in peripheral blood leukocyte is associated with mutation and cancer risk. The level of 8-OHdG in peripheral blood leukocytes can indicate a long-term response to oxidative stress rather than that in urine. Accurate identification and quantification of leukocyte 8-OHdG are essential for understanding its mechanism of formation, repair, and biological consequences. In this study, a fast and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated to detect 8-OHdG in human peripheral leukocyte. DNA in blood samples were extracted and digested, then subjected onto UHPLC–MS/MS using an isocratic elution on a Zorbax Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm). Multiple reaction monitoring (MRM) mode was adopted by using [ 15 N 5 ]-8-OHdG as an internal standard. The assay was linear over the concentration range of 1.0–100 nM with R 2 = 0.999. The accuracy for spiked samples was 90.9%–94.8%, and the intra-day precision was within 3.7%. The limit of detection (LOD) is 0.30 nM and limit of quantification (LOQ) is 1.0 nM with 5 μl of sample injection. By the analysis of human leukocyte 8-OHdG (n = 121) using the developed UHPLC–MS/MS method, it demonstrates that the level of leukocyte 8-OHdG of the cancer patients group (n = 46) is significantly higher than that of the health control (n = 75). [ABSTRACT FROM AUTHOR]

• A new molecular imprinting graphene nanocomposite (PIL-MIP/GO) was synthesized. • PIL-MIP/GO has fast mass transfer, rich adsorption mechanism, and high selectivity. • A HPLC-UV method for β-agonists was designed based on PIL-MIP/GO as PT-SPE sorbent. • The method was successfully applied to detect β-agonist in swine urine. • The analytical method was sensitive, selective, accurate, nonfatal, and economical. The illegal use of β-agonists often endangers animal-derived food safety. In this study, a selective detection method for β-agonists in swine urine was established via the combination of polymeric ionic liquid-molecularly imprinted graphene oxide-miniaturized pipette tip solid-phase extraction and high-performance liquid chromatography. It is worth noting that this method relied mainly on the designed adsorbent, which presented a rich adsorption mechanism, fast mass transfer rate, and high selectivity, and was successfully utilized in the selective extraction of β-agonists from swine urine samples. The proposed method has low LOD (0.20–0.56 ng/mL), high recovery (94.9–107.9%), and high reusability (4 times, 91.9–108.8%), which indicates its high potential as a selective, sensitive, accurate, and nonfatal method for monitoring the illegal use of β-agonists in the livestock breeding stage. [ABSTRACT FROM AUTHOR]

Highlights: [•] Different functionalized chemical groups affect the dispersibility of f-SWCNTs. [•] Insignificant effects on mitochondrial activity and ROS production in MCF7 cells by f-SWCNTs. [•] Exposure induced cell membrane damage and cell apoptosis, decreased cell adhesion. [Copyright &y& Elsevier]

This study reports the utilization of thiol-induced chemical etching of aggregation-induced emission (AIE)-active Au nanoclusters (NCs) for the facile, sensitive, and selective detection of cysteine. The AIE-active Au NCs were formed in an acidic solution containing excess Au(i)-thiolate complexes. At an acidic pH (2.0), the emission of these Au NCs was enhanced by cysteine at a concentratioin below 1 mM. However, the emission was quenched by cysteine at a high concentration, e.g., 500 mM, via the thiol-induced etching of gold, although the process occurred very slowly. Interestingly, in the absence of cysteine, increasing the solution pH enhanced the emission, while the presence of cysteine remarkably accelerated the etching-induced quenching process. The complete quenching of the emission by excess cysteine at pH 2.0 and the enhancement of the emission by the increasing pH in the absence of cysteine indicated that aurophilicity might not be involved in the AIE of the Au NCs prepared using glutathione (GSH) both as the reducing and protecting reagent. On the other hand, the etching process involved the penetration of cysteine molecules through the Au(i)-thiolate complexes, which could assemble or disassemble around the embedded Au NCs in response to the solution pH to get access to the innermost Au(0) cores. Therefore, a facile, sensitive, and selective method for the detection of cysteine was established. This method exhibited an extremely wide linear range as wide as nine orders of magnitude above the cysteine concentration, including two linear regions of the relative emission intensity of the Au NCs versus the logarithm of cysteine concentration, from 10 pM to 150 μM (correlation coefficient, 0.99851) and from 150 μM to 2 mM (correlation coefficient, 0.99866). An ultra-low limit of detection of 6.3 pM (S/N = 3) was also achieved. The developed method showed superior selectivity for cysteine relative to the 19 other natural amino acids and GSH. The method was applied for the analysis of human serum samples spiked with cysteine with satisfactory results. This study demonstrates the potential of the thiol-induced chemical etching approach as a powerful tool for studying luminescent metal NCs.

Identification and quantification of chemical DNA modifications provide essential information on genomic DNA changes, for example, epigenetic modifications and abnormal DNA lesions. In this vein, it requires to digest genomic DNA strands into single nucleosides, facilitating the mass spectrometry analysis. However, rapid digestion of such supramacromolecule DNA of several millions Daltons (molecular weight) into single nucleosides remains very challenging. Here, we constructed an immobilized benzonase capillary bioreactor and further tandemly coupled with immobilized snake venom phosphodiesterase and alkaline phosphatase capillary bioreactor to form a novel three-enzyme cascade bioreactor (BenzoSAC bioreactor). In these constructions, the chosen enzymes were immobilized onto synthetic porous capillary silica monoliths. With the tailor-made porous structure and high immobilized capacity and high digestion rate of benzonase, genomic DNA of >99.5% can be digested into single nucleosides within only 10 min when passing through the BenzoSAC bioreactor by microinjection pump. In contrast, traditional digestion requires 8-24 h. By offline coupling this benzoSAC bioreactor with liquid chromatography-tandem mass spectrometry, we detected 5-hydroxymethylcytosine, a major oxidation product of the epigenetically crucial 5-methylcytosine, in genomic DNA isolated from ladder cancer (T24) cells. The newly synthesized BenzoSAC bioreactor and the proposed mass spectrometry detection are promising for fast identification and analysis of structural modifications in DNA.

Halobenzoquinones (HBQs) are an emerging class of halogenated disinfection byproducts (DBPs) in drinking water, which raised public concerns due to potential carcinogenic effects to human bladder. Our previous work demonstrated that HBQs and hydrogen peroxide (H 2 O 2 ) together generated oxidative DNA damage via a metal-independent and intercalation-enhanced oxidation mechanism in vitro. This study further investigated the efficiency of various HBQs to induce oxidative DNA damage in T24 bladder cancer cells. Compared with T24 cells without treatment (3.1 lesions per 10 6 dG), the level of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) significantly increased by 1.4, 3.2, 8.8, and 9.2 times after treatment with tetrabromo-1,4-benzoquinone (TBBQ), terachloro-1,4-benzoquinone (TCBQ), 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ) and 2,5-dichloro-1,4-benzoquinone (2,5-DCBQ) for 24hr, respectively. Interestingly, we found that the oxidative potency of HBQs in T24 cells (2,5-DCBQ≈2,6-DCBQ>TCBQ>TBBQ) is inconsistent with that of in vitro dsDNA oxidation (TCBQ>TBBQ>2,5-DCBQ>2,6-DCBQ), suggesting HBQs induce oxidative lesions in cellular genomic DNA probably involved with a complex mechanism., (Copyright © 2017. Published by Elsevier B.V.)

Structure-based DNA modification analysis provides accurate and important information on genomic DNA changes from epigenetic modifications to various DNA lesions. However, genomic DNA strands are often required to be efficiently digested into single nucleosides. It is an arduous task because of the involvement of multiple enzymes with different catalytic acitivities. Here we constructed a three-enzyme cascade capillary monolithic bioreactor that consists of immobilized deoxyribonuclease I (DNase I), snake venom phosphodiesterase (SVP), and alkaline phosphatase (ALPase). By the use of this cascade capillary bioreactor, genomic DNA can be efficiently digested into single nucleosides with an increasing rate of ∼20 folds. The improvement is mainly attributed to dramatically increase enzymatic capacity and activity. With a designed macro-porous structure, genomic DNA of 5-30 Kb (∼1.6-10 million Daltons) can be directly passed through the bioreactor simply by hand pushing or a low-pressure microinjection pump. By coupling with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we further developed a sensitive assay for detection of an oxidative stress biomarker 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in DNA. The proposed three-enzyme cascade bioreactor is also potentially applicable for fast identification and quantitative detection of other lesions and modifications in genomic DNA.

17α-Ethinylestradiol (EE2) is the representative of environmental estrogens. Although EE2 can interact with some engineered nanoparticles (NPs), little is known about the bioactivity of NP-associated EE2 in organisms. In this study, we investigated the combined effects of the co-exposed carboxyl-functionalized single-walled carbon nanotubes (cf-SWCNTs) and EE2 in the human breast adenocarcinoma cell line (MCF-7 cells), focusing on the cytotoxicity and bioactivity. There were no significant differences in mitochondrial activity, membrane damage, and cell apoptosis when exposed to cf-SWCNTs with and without adsorbed EE2. However, the bioactivity of adsorbed EE2 on cf-SWCNTs was significantly inhibited. The calculated effective concentration of EE2 in cultured cells showed that less than 0.2% of the total adsorbed EE2 was released, indicating that most EE2 was retained on the cf-SWCNTs during cellular exposure. Furthermore, there were no obvious changes in the bioactivity of adsorbed EE2 in the culture medium containing 5-20% fetal bovine serum (FBS), even up to 10 days of incubation, indicating that the adsorbed EE2 on cf-SWCNTs is highly stable in the cell culture medium. These results mark a promising possibility for EE2 to be adsorbed by cf-SWCNTs in environmentally relevant settings and thereby influenced its toxicity and biological fate. This is also tempting for future studies involving risk assessment ways for association between NPs and contaminants in the environment.

Polyvinyl pyrrolidone polymer (PVP) has been widely applied in biological and medical fields. A few in vitro studies indicated that PVP might cause toxicity. However, the underlying mechanism is poorly understood. In this work, we found that PVP directly induced strand breakages of various DNA molecules, implicating a cleavage activity. Moreover, reactive oxygen species (ROS) scavenging analysis shows that DNA cleavage activity of PVP is not related to ROS-induced oxidation. As revealed by gel electrophoresis and liquid chromatography/mass spectrometry analysis, the major cleavage products of DNA were identified as two purine bases, guanine and adenine, suggesting that PVPs have a novel depurination activity. The selective depurination and DNA cleavage activity of PVPs were further confirmed by studying the interaction of PVP with four nucleosides and four well-designed oligodeoxynucleotides probes containing specific nucleotides. This study may provide insights into PVP-DNA interactions and resultant genotoxicity and may also open a new way for DNA study.

The toxicity of NPs is not well characterized in terms of their size. In particular, the size-based toxicity of fullerene (C(60)) remains an issue because of a lack of C(60) NPs with a well-controlled size. In this work, six fractions of the nano-C(60) aggregates (nC(60)) with different size distribution were prepared by a simple differential centrifugation. By using these nC(60) fractions, we demonstrate the size-dependent inhibition of DNA polymerase and reduced-size enhanced cytotoxicity. Above all, we found that nC(60) NPs with smaller size may have higher toxicity potency. These size-dependent effects were observed at the high exposure doses (4-6 mg/L). Interestingly, at 20-times lower and noncytotoxic doses, the size-dependent effect can be indicated by apoptosis-related fluorescent protein fused Bax translocation. Considering the toxicity of NPs is often ignored in the traditional end-point analysis for cytotoxicity when the exposure dose is low, the findings presented here will assist in the evaluation of the size-dependent cytotoxicity and dose-response relationships of toxicity mediated by nC(60) NPs at low doses.

A capillary monolithic bioreactor of snake venom phosphodiesterase (SVP) was constructed to generate different single-nucleotide mass ladders of oligodeoxynucleotides for mass spectrometry (MS)-based sequencing by immobilization. The immobilization of SVP in the porous silica monolith significantly enhances its stability for prolonged and repeated applications. The constructed capillary bioreactor has the advantages of handling (sub)microliter DNA samples and having good permeability. Benefiting from its good permeability, DNA solutions can be directly injected into the sequential digestion bioreactor simply by hand pushing or a low-pressure microinjection pump. Moreover, the immobilization of SVP facilitates the elimination or repression of the metal adducts of oligodeoxynucleotides, improving the analytical performance of MS sequencing. By the application of capillary bioreactor of immobilized SVP, the sequence-specific modification of single-stranded oligodeoxynucleotide induced by a ubiquitous pollutant acrolein (Acr) was identified, demonstrating its promising applications in identification of sequence-specific damage, which may further our understanding of DNA damage caused mutagenesis.

Bisphenols (BPs) are potential endocrine-disrupting chemicals that may adversely affect human health and wildlife. The complexity of matrix encountered in real-world samples renders screening of trace BPs a formidable challenge. The present study highlighted the potential of molecularly imprinted solid-phase extraction (MISPE) for selective detection of trace bisphenols and their halogenated analogues in surface water. The template bleeding was observed at parts-per-billion levels, deteriorating the accuracy and precision of BPs quantification. To surmount this problem, a dummy MISPE strategy was proposed, in which bisphenol E (BPE) was selected as a dummy template for molecularly imprinted polymer (MIP) synthesis. Coupling this MISPE strategy with chromatographic analysis, a dummy MISPE-HPLC method was established. The linearity, precision, limit of detection (LOD) and recovery were then validated. The linearity of the calibration curve for each BP was observed over the range of 20-2000 ng L-1 (r > 0.998). LOD for each bisphenol was measured as low as 2.5-5.0 ng L-1. This technique was applied to simultaneous screening of BPs in the Qinghe River, and five bisphenols were found within the concentration range of 0-224 ng L-1 in river samples. The designed dummy MIP was superior to the commercial sorbents with regard to the selectivity, cross-reactivity, matrix removal efficiency and reusability. These merits enabled the applications of dummy MISPE for selective extraction and sensitive screening of BPs in environmental water samples. This method also provided a promising tool for monitoring the occurrence, distribution and fate of BPs in surface water.

An important property of C(60) in aquatic ecotoxicology is that it can form stable aggregates with nanoscale dimensions, namely nC(60). Aggregation allows fullerenes to remain suspended for a long time, and the reactivity of individual C(60) is substantially altered in this aggregate form. Herein, we investigated the interaction of nC(60) and human serum album (HSA) using the methods of fluorescence, fluorescence dynamics, circular dichroism (CD), and site marker competitive experiments. We proposed a binding model consistent with the available experimental results for the interactions of nC(60) with HSA. During the interaction process, the structure and conformation of HSA were affected, leading to functional changes of drug binding sites of HSA.

Aristolochic acid (AA) is a known nephrotoxin and potential carcinogen, which can form covalent DNA adducts after metabolic activation in vivo and in vitro. A simple method for preparation and characterization of aristolochic acid-DNA adducts was developed. Four AA-adducts were synthesized by a direct reaction of AAI/AAII with 2'-deoxynucleosides. The reaction mixture was first cleaned-up and pre-concentrated using solid phase extraction (SPE), and further purified by a reversed-phase high performance liquid chromatography (HPLC). By the application of developed SPE procedure, matrices and byproducts in reaction mixture could be greatly reduced and adducts of high purity (more than 94% as indicated by HPLC) were obtained. The purified AA-DNA adducts were identified and characterized with liquid-electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-Q-TOF-MS/MS) and LC-Diode array detector-fluorescence (LC-DAD-FL) analysis. This work provides a robust tool for possible large-scale preparation of AA-DNA adduct standards, which can promote the further studies on carcinogenic and mutagenic mechanism of aristolochic acids.

We have found a new way, a metal-organic coordination gel template method, to generate polymer monoliths with an essentially macropore size, in which the pores are organized spontaneously into continuous networks; furthermore, the resulting macroporous monoliths have potential applications in high-throughput and high-efficiency separation of proteins.

An on-line SPE-HPLC method, using a monolithic poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (poly(GMA-EDMA)) based weak cation-exchange (WCX) column, was developed for simultaneous determination of alpha1-adrenergic receptor antagonists in human plasma. The monolithic WCX column was prepared by an in-situ polymerization protocol and modified stepwise with ethylenediamine and chloroacetic acid. On connecting this column to an injection valve, an on-line SPE protocol could be established for removal of matrices (mainly proteins and lipids) and preconcentration of four alpha1-adrenergic receptor antagonists in human plasma. This method was validated and then used for determination of terazosin, alfuzosin, prazosin, and doxazosin in clinical plasma samples. For all analytes, each calibration curve was found to be linear over a range of 0.005-5 microg/mL (R>0.997), and the limit of detection for each analyte was 0.5 ng/mL. Recovery (>80%) and precision (RSD<15%) for inter- and intra-day assay were tested at three concentration levels of each analyte and showed acceptable results for quantitative assay. Real samples from hypertensive patients were monitored and results were in agreement with those of the conventional liquid-liquid extraction-HPLC method. Furthermore, due to its good permeability and biocompatibility, the monolithic WCX sorbent could be reused more than 300 times. The proposed method was especially appropriate for multi-analyte monitoring in plasma samples.