Separation of cytokinin isomers with a partial filling-micellar electrokinetic chromatography-mass spectrometry approach

A new method based on partial filling-MEKC (PF-MEKC) directly coupled to ESI-MS was developed for the simultaneous separation and determination of 13 structurally similar cytokinins, including various geometric and positional isomers of cytokinins. On the basis of the resolution of the neighboring isomer peaks, different parameters (i.e., pH and concentration of buffer, surfactant concentrations, length of the injected micellar plug, organic modifier, and applied separation voltage) were optimized to achieve a satisfactory PF-MEKC separation. Under optimum conditions, the separation of 13 cytokinin standards was accomplished within 25 min. MS/MS with multiple reaction monitoring detection was carried out to obtain sufficient selectivity. PF-MEKC-MS/MS allowed for the direct identification and confirmation of the cytokinins present in banana (Musa spp.) pulp sample after extraction and purification. Finally, trans-zeatin riboside (ZR) and trans-zeatin (Z) were unambiguously identified in banana pulp. It is anticipated that the current PF-MEKC-MS method can be applied to analyze cytokinins in a wide range of biological samples.     

Immunoassay for native enzyme quantification in biological samples

In order to detect low levels of enzyme activity, specifically glucose oxidase, in biological samples, an immunoenzymatic assay was developed since currently available methods could not be used because of either their lack of sensitivity or the conditions prevailing in our samples: turbidity of the medium, presence of redox systems other than glucose oxidase, and high concentration of proteins. The principle of the method is to coat a polystyrene surface with a fragment Fc-specific anti-IgG, then with an antibody directed against the looked-for enzyme, which is simultaneously the antigen and the enzyme activity required for immunoenzymatic detection. We applied this concept to biological samples after glucose oxidase administration to mice. This method achieves specificity and sensitivity (20 ng/mL or 1 ng) with samples of biological origin. No marker is needed since the antigen itself possesses an enzyme activity. This method, which requires a small sample volume (50 ΜL, 20 ΜL, if necessary), can be extended easily to the many enzymes currently used as markers. It could also be applied to the native enzymes of medical interest for which antibodies and a colorimetric reaction are available.     

Spatially addressable protein array: ssDNA-directed assembly for antibody microarray

Protein microarray offers a means for high-throughput profiling of cellular proteins to provide insights into the mechanisms of biological processes. This study describes the design and fabrication of a robust platform, spatially addressable protein array (SAPA), by exploring the specificity of ssDNA hybridization for self-assembly of semi-synthetic ssDNA-antibody conjugates which capture antigens from complex biological samples. This approach does not involve the direct immobilization of antibodies nor antigen, but instead captures the target antigens in the solution phase followed by self-directed assembly of the complex onto the surface. In an effort to optimize the platform, the effects of surface chemistry, nonspecific protein adsorption, facile preparation, and purification of ssDNA-conjugated antibody and capture of the antigen from a complex biological sample such as cell lysate were examined. This platform allowed antigen detection in cell lysate with high sensitivity (1 pM). The method described herein can be extended to the high-throughput detection of other interacting molecules in solution phase and their subsequent assembly onto any substrate.     

Enzyme-linked immunosorbent assay for the determination of T-2 toxin in cereals and feedstuff

A reliable enzyme-linked immunosorbent assay method was developed for the assay of T-2 toxin in cereals and feedstuff. A hapten of the T-2 toxin was synthesized, and a polyclonal antibody with high affinity and specificity was obtained after immunization of rabbits. Compared to the other ELISA methods, the assay is simple, rapid and affordable. The concentration of T-2 causing 15% inhibition is 0.01?±?0.001 ng mL^?1, which makes the method more sensitive than others. The cross-reactivity against other mycotoxins is low, except for the HT-2 toxin. Sample extraction was achieved within 3 min. The recoveries from samples including barley, wheat, corn, oat, rice and feedstuff were between 75% and 102%, and the detection limit for T-2 toxin was lower than 4 ng g^?1. The method was validated by high-performance liquid chromatography with tandem mass spectrometry detection.     

Development of a method for comprehensive and quantitative analysis of plant hormones by highly sensitive nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry

In recent plant hormone research, there is an increased demand for a highly sensitive and comprehensive analytical approach to elucidate the hormonal signaling networks, functions, and dynamics. We have demonstrated the high sensitivity of a comprehensive and quantitative analytical method developed with nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-IT-MS/MS) under multiple-reaction monitoring (MRM) in plant hormone profiling. Unlabeled and deuterium-labeled isotopomers of four classes of plant hormones and their derivatives, auxins, cytokinins (CK), abscisic acid (ABA), and gibberellins (GA), were analyzed by this method. The optimized nanoflow-LC-ESI-IT-MS/MS method showed ca. 5-10-fold greater sensitivity than capillary-LC-ESI-IT-MS/MS, and the detection limits (S/N = 3) of several plant hormones were in the sub-fmol range. The results showed excellent linearity (R² values of 0.9937-1.0000) and reproducibility of elution times (relative standard deviations, RSDs, <1.1%) and peak areas (RSDs, <10.7%) for all target compounds. Further, sample purification using Oasis HLB and Oasis MCX cartridges significantly decreased the ion-suppressing effects of biological matrix as compared to the purification using only Oasis HLB cartridge. The optimized nanoflow-LC-ESI-IT-MS/MS method was successfully used to analyze endogenous plant hormones in Arabidopsis and tobacco samples. The samples used in this analysis were extracted from only 17 tobacco dry seeds (1 mg DW), indicating that the efficiency of analysis of endogenous plant hormones strongly depends on the detection sensitivity of the method. Our analytical approach will be useful for in-depth studies on complex plant hormonal metabolism.     

New rhodamine nitroxide based fluorescent probes for intracellular hydroxyl radical identification in living cells

The synthesis, characteristics, and biological applications of a series of new rhodamine nitroxide fluorescent probes that enable imaging of hydroxyl radicals (?OH) in living cells are described. These probes are highly selective for ?OH in aqueous solution, avoiding interference from other reactive oxygen species (ROS), and they facilitate ?OH imaging in biologically active samples. The robust nature of these probes (high specificity and selectivity, and facile synthesis) offer distinct advantages over previous methods for ?OH detection.     

孔雀石绿单克隆抗体制备和酶联免疫检测方法的建立

针对孔雀石绿三苯环特征结构,设计合成1种高质量半抗原。通过偶联载体蛋白、动物免疫和细胞融合,制备出一株高质量的单克隆抗体MG-DA4-C7,亚类为IgG1,轻链为κ型。通过对包被原浓度、抗体浓度、二抗浓度的优化,建立了孔雀石绿间接竞争酶联免疫分析方法。方法半抑制浓度( IC50)为0.96μg/L,线性检测范围(IC20~C80)为0.1~8.1μg/L,LOD(IC10)为0.05μg/L,回归方程y=-0.3274lgx+0.4698(R2=0.9891)。本方法特异性高,与代谢物隐孔雀石绿交叉反应小于0.1％,与结晶紫、灿烂绿交叉反应率分别为18.1％和26.5％;实际样品添加回收率为87.3％~107.3％。本方法测定结果经HPLC-MS/MS方法确证,二者相关系数达0.999。本方法可用于鱼类等水产品中孔雀石绿残留的实际检测。     

Thallium determination in biological materials by radiochemical neutron activation analysis

Summary The determination of thallium in biological materials sometimes cause problems because of the low concentrations of this toxic element. In the present work a method is described which optimizes the parameters affecting the specificity and sensitivity of the radiochemical NAA of thallium in biological samples. High thermal neutron flux, complete decomposition of the organic matter by pressurized digestion, TlI precipitations, liquid extraction of HTlBr₄ and La(OH)₃ scavenging purification are the steps leading to the final homogeneous preparation of Tl₂CrO₄ for -activity measurement. The method was applied to various materials as bovine liver, bone and nails. Good agreement was found between certified and determined thallium concentrations of the reference material CRM 176. The chemical yield comes to about 80%, with low deviations. The sensitivity of the method is about 10^–3 g/g, the standard deviations being in the range of 3.6% (CRM 176), 14% (bovine liver), and 17% (bone). Detailed working instructions are given.     

Application of computer-assisted molecular modeling for immunoassay of low molecular weight food contaminants: A review

Immunoassay for low molecular weight food contaminants, such as pesticides, veterinary drugs, and mycotoxins is now a well-established technique which meets the demand for a rapid, reliable, and cost-effective analytical method. However, due to limited understanding of the molecular structure of antibody binding sites and antigenic epitopes, as well as the intermolecular binding forces that come into play, the traditional ‘trial and error’ method used to develop antibodies still remains the method of choice. Therefore, development of enhanced immunochemical techniques for specific- and generic-assays, requires new approaches for antibody design that will improve affinity and specificity of the antibody in a more rapid and economic manner. Computer-assisted molecular modeling (CAMM) has been demonstrated to be a useful tool to help the immunochemist develop immunoassays. CAMM methods can be used to help direct improvements to important antibody features, and can provide insights into the effects of molecular structure on biological activity that are difficult or impossible to obtain in any other way. In this review, we briefly summarize applications of CAMM in immunoassay development, including assisting in hapten design, explaining cross-reactivity, modeling antibody-antigen interactions, and providing insights into the effects of the mouse body temperature on the three-dimensional conformation of a hapten during antibody production. The fundamentals and theory, programs and software, limitations, and prospects of CAMM in immunoassay development were also discussed.     

Determination of prostaglandins and thromboxane in whole blood by high-performance liquid chromatography with fluorimetric detection

A highly sensitive and specific assay for the quantitation of prostaglandins (PGs) such as PGE1, PGE2, PGF1 alpha, PGF2 alpha, 6-keto-PGF1 alpha, and including thromboxane B2, is described. The method involves the addition of PGF1 alpha and PGE1 as the internal standards, extraction from whole blood and purification by silica gel column chromatography. Following conversion into the methoximes, purification by reversed-phase chromatography and esterification with panacyl bromide, samples are analysed by high-performance liquid chromatography with fluorimetric detection. The lower limit of detection of the eicosanoids 6-keto-PGF1 alpha, thromboxane B2 and PGF2 alpha in blood is ca. 50 pg/ml and that of PGE2 is 100 pg/ml. Assay linearity is demonstrated over a range from 60 pg to 60 ng of eicosanoid injected. The method allows simultaneous assessment of prostaglandins and thromboxane extracted from complex biological fluids at picogram levels.     

Development of a fluorescence polarization immunoassay for the detection of melamine in milk and milk powder

A fluorescence polarization immunoassay (FPIA) based on a polyclonal antibody was developed for the determination of melamine in milk. To obtain an antibody with improved sensitivity and specificity, 6-hydrazinyl-1,3,5-triazine-2,4-diamine was coupled to bovine serum albumin and used as the immunogen for the rabbit immunization. Three fluorescein-labeled melamine tracers with different structures and spacer bridges were synthesized. The structural effect of the tracers on the assay characteristics was investigated. 6-(4,6-Diamino-1,3,5-triazin-2-ylamino)-N-(2-(3-(3′,6′-dihydroxy-3-oxo-2,3-dihydrospiro[indene-1,9′-xanthene]-5-yl)thioureido)ethyl)hexanamide demonstrated better sensitivity than 5-(2-(4,6-diamino-1,3,5-triazin-2-yl)hydrazinecarbothioamido)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid and 3-(4,6-diamino-1,3,5-triazin-2-ylthio)-N-(2-(3-(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-yl)thioureido)ethyl)propanamide. The limit of detection (10% inhibition) of the FPIA was 9.3 ng mL^-1 and the IC₅₀ (50% inhibition) value was 164.7 ng mL^-1. The antibody in the FPIA showed 21.2% cross-reactivity to the fly-killing insecticide cyromazine, but had no cross-reactivity to other natural structurally related compounds. Recoveries, measured in spiked milk and milk powder samples, ranged from 79.4 to 119.0%. Milk samples fortified with melamine were analyzed by this method and confirmed by high-performance liquid chromatography–mass spectrometry. Excellent recoveries and correlation with spiked levels were observed, suggesting that this immunoassay could be applied to the screening of melamine residues in milk and milk powder after a simple dilution procedure.      

A highly sensitive enzyme immunoassay for evaluation of 2′-deoxycytidine plasma level as a prognostic marker for breast cancer chemotherapy

A highly sensitive competitive enzyme immunoassay (EIA) has been developed and validated for the determination of the plasma level of 2′-deoxycytidine (dCyd), the potential prognostic marker for breast cancer chemotherapy. This assay employed a monoclonal antibody that recognizes dCyd with a high specificity, and 5′-succinyl-dCyd (5′sdCyd) conjugate of bovine serum albumin (5′sdCyd-BSA) immobilized onto microplate wells as a solid phase. The assay involved a competitive binding reaction between dCyd, in plasma sample, and the immobilized 5′sdCyd-BSA for the binding sites of the anti-dCyd antibody. The bound antibody was quantified with horseradish peroxidase-labeled anti-immunoglobulin second antibody and 3,3′,5,5′-tetramethylbenzidine as a peroxidase substrate. The concentration of dCyd in the sample was quantified by its ability to inhibit the binding of the antibody to the immobilized 5′sdCyd-BSA and subsequently the color formation in the assay. The assay limit of detection was 8 nM and the effective working range at relative standard deviations (R.S.D.s) of ≤10% was 20-800 nM. No cross-reactivity from the structurally related nucleobases, nucleosides, and nucleotides was observed in the proposed assay. Mean analytical recovery of added dCyd was 98-100 ± 3.2-8.2%. The precision of the assay was satisfactory; R.S.D. was 3.4-4.2 and 4.3-8.9% for intra- and inter-assay precision, respectively. The proposed EIA was compared favorably with HPLC method in its ability to accurately measure dCyd spiked into plasma samples. The analytical procedure is convenient, and one can analyze 200 samples per working day, facilitating the processing of large-number batch of samples. The proposed EIA is expected to contribute in further evaluation of dCyd as a prognostic marker for breast cancer chemotherapy and elucidation of the role of dCyd in various biological and biochemical systems.     

荧光偏振免疫分析方法快速检测沙拉沙星残留

以异硫氰酸荧光素(FITC)标记沙拉沙星合成荧光标记物,采用薄层色谱法提纯,优化了反应时间、标记物和抗体的工作浓度,建立了沙拉沙星的快速荧光偏振免疫分析法( FPIA).本方法测定沙拉沙星在缓冲液中的半数抑制浓度(IC50)为43.2 μg/L;检测范围为5.7～327 μg/L,可以达到国家规定的动物性食品中兽药最高残留限量(80 μg/kg)的要求.本研究考察了FPIA测定沙拉沙星的动力学过程及对其它4种喹诺酮类药物的交叉反应.结果表明,环丙沙星、恩诺沙星、加替沙星及氧氟沙星的交叉反应率分别为3.3％,1.8％,1.7％和0.7％.在牛奶和猪尿中沙拉沙星的回收率分别在71％～94％和74％～102％之间.本方法操作简单快捷,整个检测过程只需5 min、而且灵敏度较高、特异性强,适用于动物性食品中沙拉沙星残留的快速筛选检测.     

马鲛鱼抗氧化肽的纳流液相色谱法分离与筛选

在海洋天然产物中,马鲛鱼是一种重要的高活性抗氧化肽生物源,具有极高的加工附加值。由于鱼体组织的复杂性,活性抗氧化肽成分的提取和筛选对样品制备和分离技术提出了挑战。使用不同蛋白酶对鱼体组织进行酶解时,所获得的活性肽结构及功能活性会有显著的差别。为了获得高活性的抗氧化肽,该研究分别考察了风味蛋白酶、胰蛋白酶、酸性蛋白酶、中性蛋白酶、碱性蛋白酶5种蛋白酶的酶解效果。以二苯代苦味肼基自由基(DPPH·)、羟自由基(·OH)清除率和水解度(DH)为指标,筛选最优水解酶。结果表明,胰蛋白酶酶解液清除DPPH·和·OH能力最强,清除率分别达到88.93%±0.82%和53.09%±0.73%。在单因素试验的基础上,以DPPH·清除率为响应值,以加酶量、酶解温度和时间为函数,进行了三因素三水平响应面试验,获得水解度23.66%、DPPH·清除率93.78%以及·OH清除率62.59%的最优制备条件。纳流液相色谱具有低样品量、低溶剂消耗和高效等优势。为筛选出适合于马鲛鱼内脏抗氧化肽分离分析的固定相,该研究使用1∶1000分流比的纳流液相平台,分别使用反相C18柱(15 cm×100 μm, 5 μm, 30 nm)和强阳离子交换柱(15 cm×100 μm, 5 μm, 100 nm)进行分离,收集、冻干并评测了各组分的抗氧化能力。结果表明,强阳离子交换固定相更适合于马鲛鱼内脏抗氧化肽的分离纯化,并筛选出1个强活性抗氧化肽组分。该组分DPPH·清除力的半抑制浓度(IC₅₀)为0.672±0.051 mg/mL,与纯化前相比提高了13.6倍。该研究报道了纳流液相色谱在海洋天然产物源抗氧化肽分离分析中的应用,并证明了其在活性抗氧化肽成分筛选中的有效性和良好的应用前景。     

A novel method for purification of biologically active C-terminal fragment of human recombinant anti-mullerian hormone

Anti-mullerian hormone (AMH) is one of the least studied members of transforming growth factor beta superfamily showing pro-apoptotic activity against cells positive for hormone type II receptor overexpressed by malignant cells in many cancer cases. Here, we propose an improved method for isolation of recombinant C-terminal AMH fragment (C-rAMH) to obtain homogeneous preparations of this protein with high biological activity. In contrast to our previously developed C-rAMH purification technology based on reversed-phase HPLC, the key stage of the new approach is hydrophobic interaction chromatography using Toyopearl Butyl-650S resin performed under more benign conditions. This modification of the previously developed method allowed highly purified C-rAMH to be obtained that is characterized by twice the specificity estimated as the ability to bind to the recombinant analog of AMH type II receptor and by significantly higher biological activity, that is, the ability to induce the death of target cells. Thus, we made the purification technology even more cost-effective and suitable for the production of drug forms based on C-rAMH.     

Development and validation of a highly sensitive ELISA for the determination of pharmaceutical indomethacin in water samples

The development and validation of a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of pharmaceutical indomethacin in water samples was presented. The immunogen and coating antigen were prepared by covalently linking indomethacin to bovine serum albumin and ovalbumin by anhydride ester method. Two rabbits were immunized by standard immunization processes and the superior antibody was characterized in terms of sensitivity, specificity, precision, accuracy and stability. Under optimal experimental conditions, the standard curve was constructed in the concentration range of 0.01-10 ng/mL. For 10 consecutive standard curves run in 2 weeks, IC₅₀ value (the concentration of analyte producing 50% of inhibition) were found within 0.10-0.25 ng/mL, and the detection limit (DL) at a signal-to-noise ratio of 3 (S/N = 3) was about 0.01 ng/mL. The antiserum recognized acemetacin, a precursor of indomethacin with 92.3% cross-reactivity, while the cross-reactivity values of antiserum with other tested compounds were very low. From the spiking experiments, the recoveries were found within 98-123%. The ELISA was applied for the determination of indomethacin in different water samples and the results were confirmed by conventional HPLC. The correlation coefficient of 0.988 was obtained, demonstrating a good correlation of ELISA with HPLC.     

Quantitative analysis of cytokinins in plants by liquid chromatography–single-quadrupole mass spectrometry

A sensitive method for the quantitative analysis of all natural isoprenoid cytokinins in plant material by electrospray single-quadrupole mass spectrometry is presented. A baseline chromatographic separation of 20 non-derivatised naturally occurring cytokinins has been developed. Precise analyses of O-glucoside and ribonucleotide fractions were also performed by the high-performance liquid chromatography–mass spectrometry (HPLC–MS) but run separately from the basic cytokinin metabolites. Using post-column splitting, the flux from narrow-bore (2.1 mm i.d.) reversed-phase liquid chromatography column was simultaneously introduced into the diode array and mass detector. Optimal conditions, including final flow rate, desolvation temperature, desolvation gas flow, capillary and cone voltage for effective ionisation in the electrospray ion source were found. When low cone voltage (20 V) was applied, all studied cytokinins were determined in aqueous methanol as dominant quasi-molecular ions of [M+H]⁺ with limits of detection ranging between 10 and 50 fmol. For routine analysis a linearity range between 25 (75) fmol and 100 pmol was obtained. Developed liquid chromatography–mass spectrometry (LC–MS) method in selective ion monitoring mode was employed to quantify cytokinin species in tobacco BY-2 suspension culture and poplar leaves (Populus×canadensis Moench, cv Robusta).

Purified plant cell (BY-2) and plant tissue (poplar leaves) extracts were obtained by using two different ion-exchange chromatography steps, in combination with immunoaffinity purification using a broad-spectrum monoclonal anti-cytokinin antibody. The antibody strongly recognises the presence of N⁶-substituent on purine skeleton and thus does not bind adenine and related compounds. The presence of authentic cytokinins in the extracts quantified by LC–MS was further verified by enzyme-linked immunosorbent assays (ELISAs) with prior LC preparation. The combination of liquid chromatography–single-quadrupole mass spectrometry with immunoaffinity chromatography offers an efficient and elegant method for detection and quantification of cytokinin metabolites.     

Development of a sensitive, rapid, biotin-streptavidin based chemiluminescent enzyme immunoassay for human thyroid stimulating hormone

A highly sensitive "two-site" chemiluminescent immunoassay specific for human thyroid stimulating hormone (TSH) was developed. The signal amplification was achieved via a biotin-streptavidin system (BSAS). The HRP-luminol-H(2)O(2) chemiluminescent system with high sensitivity was chosen as the detection system. Biotinylated anti-TSH monoclonal antibody (MAb) and HRP-labeled streptavidin were first synthesized. Then the signal amplification was achieved through the interaction between the biotinylated anti-TSH MAb and the HRP-streptavidin conjugate. The light intensity developed was in proportion to the TSH present in the samples. The assay showed little cross-reactivity with three other glycoprotein hormones (human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle stimulating hormone (FSH)) due to the high specificity of the antibody. The working range for human thyroid stimulating hormone was 0.1-40 mU L(-1). Both the intra-assay and inter-assay coefficients of variation were less than 10% for the BSAS based chemiluminescent enzyme immunoassay (CLEIA). The proposed assay had a sensitivity of 0.01 mU L(-1) which was 10-fold higher than the HRP-MAb conjugate based TSH immunoassay. Thus the higher sensitivity facilitated the clinical testing for thyroid states. The effects of several reaction parameters, such as incubation time, temperature, and reaction volume of the method, were also studied. This method has been successfully applied to the evaluation of TSH in human serum. Compared with the commercial enzyme chemiluminescent immunoassay, the correlation was satisfied.     

Application of in-gel protease assay in a biological sample: characterization and identification of urokinase-type plasminogen activator (uPA) in secreted proteins from a prostate cancer cell line PC-3

A considerable problem in proteomics is to separate and identify functional proteins that participate in specific biological processes. To expedite the analysis of active proteases, we have developed a substrate-specific, sensitive in-gel trypsin activity assay after two-dimensional (2-D) separation in a sodium dodecyl sulphate (SDS)-polyacrylamide gel [22]. Using this method, we detected and characterized Arg-specific protease activity in the secreted protein sample of a prostate cancer cell line, PC-3, in 1-D and 2-D gels. Mass spectrometry (MS) identified the protease as urokinase-type plasminogen activator (uPA). Western blotting using anti-uPA antibody and protease inhibition tests confirmed the identification. Since no antibody was involved in the procedure, the result clearly demonstrates the feasibility of this method for identifying novel proteases in biological samples.     

Development of a validated HPLC method for the determination of four 1,4-benzodiazepines in human biological fluids

A simple and sensitive HPLC method was developed and validated for the determination of four frequently prescribed 1,4-benzodiazepines: alprazolam (ALP), bromazepam (BRZ), diazepam (DZP), and flunitrazepam (FNZ). Separation was achieved on an Inertsil C8 analytical (250 mm x 4 mm, 5 microm) column, after selective extraction of benzodiazepine drugs from biological matrices by means of SPE. Isocratic elution was performed with a mobile phase consisting of CH3COONH4, 0.05 M CH3OH, and CH3CN (33:57:10 by volume). Quantification was performed at 240 nm with mefenamic acid (6 ng/microL) as the internal standard. DSC-18 Supelco cartridges provided high absolute recoveries (81-115%). The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, and sensitivity. Repeatability (n = 8) and between-day precision (n = 8) revealed RSD <12%. Recoveries from biological samples ranged from 81.2 to 115%. The detection limit of the method was calculated as 3.3-10.2 ng in blood plasma and 2.6-12.6 ng in urine for 20 microL injection volume. The method was applied to spiked biological matrices. Moreover, the method was applied to real samples of urine after an oral administration.