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.     

Validation of an enzyme-linked immunosorbent assay (ELISA) for the determination of 4-nonylphenol and octylphenol in surface water samples by LC-ESI-MS

4-Nonylphenol (NP) and octylphenol (OP) were measured by direct ELISA in both laboratory-fortified and surface water samples collected monthly from 10 rivers. In this procedure, samples were concentrated by solid phase extraction (SPE) using Lichrolut RP-18 sorbent with good recoveries obtained for both LC-MS and ELISA, giving a low level of detection (LOD) at the range of low μg L⁻¹ and good reproducibility. Analysis of 40 surface water samples demonstrated that the ELISA may be a useful screening tool for the determination of the alkylphenols in surface water matrices. The concentration of NP and OP in surface waters ranged from 0.11 to 6.58 μg L⁻¹. A good correlation of results obtained by ELISA and LC-MS within the concentration range of 0.08-6.86 μg L⁻¹ was found in the river samples [R² = 0.924, n = 39]. The influence of various factors on assay determination was also discussed.     

Square wave anodic stripping voltammetric determination of Pb2+ using acetylene black paste electrode based on the inducing adsorption ability of I-

In the study, we developed a simple, rapid and sensitive method for the determination of tiopronin (TP) in human plasma, which was based on derivatization with p-bromophenacyl bromide (p-BPB) followed by liquid-liquid extraction and reverse-phase HPLC-UV detection. For the first time, the p-BPB was introduced into the derivatization of TP. The thiol group of TP was trapped with p-BPB to form a TP-p-BPB adduct, which can be very suitable for UV detection. From acidified plasma samples, the derivatized TP was extracted with 5 mL dichloromethane. Effective chromatographic separation was achieved using a C18 column (DIAMONSIL 150 mm × 4 mm i.d., 5 μm) based on an acetonitrile-water-trifluoroacetic acid (40:59.88:0.12, v/v/v) elution at a flow-rate of 1 mL/min. The IS and the derivatized TP were detected at 263 nm. No endogenous substances were found to interfere. The limit of quantification for derivatized TP (TP-p-BPB) in plasma was 40 ng/mL. The calibration curve for the derivatized TP showed linearity in the range 0.04-4 μg/mL with a regression coefficient corresponding to 0.9991 and the coefficient of the variation of the points of the calibration curve being lower than 10%. Extraction recoveries of the derivatized TP in plasma were greater than 72%. The method was suitably validated and successfully applied to determination of TP in human plasma samples.     

Quantification of cephalexin as residue levels in bovine milk by high-performance liquid chromatography with on-line sample cleanup

A multidimensional, selective and precise high performance liquid chromatography (HPLC) method based on direct injection of biological samples has been developed for the determination of cephalexin in skimmed and whole bovine milk. The cephalosporin antibiotic was extracted from bovine milk using an octyl restricted access medium bovine serum albumin column (C₈-RAM-BSA) and analyzed on an octadecyl column using phosphate buffer (pH 7.5, 0.01 M): ACN (92:8, v/v) and ultraviolet detection at 260 nm. The calibration graph was linear in the concentration range of 25-1600 ng/mL and this is in accordance with the tolerance level of 100 ng/mL set by the FDA (Food and Drug Administration) and EU (European Union) for cephalexin as residue in bovine milk. The intra- and inter-day coefficients of variation for the replicate analysis at the quality control levels were less than 15% while the transfer efficiency was in the range of 90.2-92.3%. The limits of detection and quantification were 10 and 20 ng/mL, respectively.     

Analysis of commercial beverage products by size exclusion chromatography coupled with UV-vis absorbance detection and dynamic surface tension detection

Multidimensional analysis of instant coffee and barley beverage samples using size exclusion chromatography (SEC) combined with a dynamic surface tension detector (DSTD) and a UV-vis absorbance detector (UV) is reported. A unique finding of this study was the action of the tetrabutylammonium (TBA) cation as a modifying agent (with bromide as the counter anion) that substantially increased the surface pressure signal and sensitivity of many of the proteins in the chromatographically separated samples. The tetrabutylammonium bromide (TBAB) enhancement of the surface pressure signal was further investigated by studying the response of 12 commercial standard proteins (α-lactalbumin, β-lactoglobulin, human serum albumin (HSA), albumin from chicken egg white (OVA), bovine serum albumin (BSA), hemoglobin, α-chymotrypsinogen A, cytochrome C, myoglobin, RNase A, carbonic anhydrase, and lysozyme) in buffer performed using flow injection analysis (FIA) coupled with the DSTD with and without various concentrations of TBAB. The FIA-DSTD data show that 1 mM TBAB enhances sensitivity of HSA detection, by lowering the limit of detection (LOD) from 2 mg/mL to 0.1 mg/mL. Similarly, the LOD for BSA was reduced from 1 mg/mL to 0.2 mg/mL. These FIA-DSTD experiments allowed the detection conditions to be optimized for further SEC-UV/DSTD experiments. Thus, the SEC-UV/DSTD system has been optimized and successfully applied to the selective analysis of surface-active protein fractions in a commercial instant coffee sample and in a soluble barley sample. The complementary selectivity of using the DSTD relative to an absorbance detector is also demonstrated.     

Electrochemical immunoassay of hepatitis B surface antigen by the amplification of gold nanoparticles based on the nanoporous gold electrode

We describe herein the combination of electrochemical immunoassay using nanoporous gold (NPG) electrode with horseradish peroxidase (HRP) labeled secondary antibody-gold nanoparticles (AuNPs) bioconjugates for highly sensitive detection of protein in serum. The electroactive product of o-phenylenediamine (OPD) oxidized with H₂O₂ catalyzed by HRP was reduced in the Britton-Robinson (BR) buffer and the peak current of which was used to determine the concentration of antigen (Ag) in the analyte. The active surface area of NPG electrode was larger than that of a bare flat one. The presence of AuNPs enhanced the immobilized amount of HRP labeled antibody (Ab), which improved the sensitivity of the immunoassay when used as the secondary antibodies. As a result of these two combined effects, the sensitivity of the immunoassay for the determination of target protein was increased significantly. Using hepatitis B surface antigen (HBsAg) as a model, we demonstrate a dose response in the range of 0.01-1.0 ng/mL with a detection limit of 2.3 pg/mL. Analytical results of several human serum samples obtained using the developing technique are in satisfactory agreement with those given by enzyme-linked immune-absorbent assays (ELISA). In addition, the technique was about 100 times more sensitive in the detection of HBsAg than ELISA. All these demonstrated the feasibility of the present immunoassay method for clinical diagnosis.     

An in-tube SPME device for the selective determination of chlorophyll a in aquatic systems

We report a new device for the estimation of the content of chlorophyll a pigment in water samples as an indicator of water quality. The extraction of the pigment from water was also optimized. 10 mL of water was filtered through a nylon filter (45 μm pore size and 13 mm of diameter), after the chlorophylls were dissolved by immersing the filter in 1 mL of a low non-hazardous solvent as ethanol. An in-valve in-tube SPME device coupled to capillary liquid chromatography with diode array detection was designed. A capillary column of 70 cm in length (0.32 mm i.d. coated with 5% diphenyl-95% polydimethylsiloxane, 3 μm coating thickness) was used as the loop of the injection valve for preconcentration and a Zorbax SB C₁₈ (SiO₂-based) 150 mm × 0.5 mm i.d., 5 μm column (Agilent) was used as analytical column. The achieved detection limit was 0.05 μg L⁻¹ and the working range of concentrations was 0.1-1 μg L⁻¹. % RSD values between 2 and 11 were obtained. Chlorophyll a in several water matrices was determined with good results in presence of other pigments such as chlorophyll b, pheophytin a and pheophytin b.     

A novel sorptive extraction method based on polydimethylsiloxane frit for determination of lung cancer biomarkers in human serum

In this study, a porous polypropylene frit was coated with polydimethylsiloxane (PDMS) as extraction medium, based on the home-made PDMS-frit, a rapid, simple and sensitive sorptive extraction method was established for analysis of potential biomarkers of lung cancer (hexanal and heptanal) in human serum samples. In the method, derivatization and extraction occurred simultaneously on the PDMS-frit, then the loaded frit was ultrasonically desorbed in acetonitrile. Polymerization, derivatization–extraction and desorption conditions were optimized. Under the optimal conditions, satisfactory results were gained, a wide linear application range was obtained in the range of 0.002–5.0 μmol L⁻¹ (R > 0.997) for two aldehydes, the detection limits (S N⁻¹ = 3) were 0.5 nmol L⁻¹ for hexanal and 0.4 nmol L⁻¹ for heptanal. The relative standard deviations (RSDs, n = 5) of the method were below 7.9% and the recoveries were above 72.7% for the spiked serum. All these results hint that the proposed method is potential for disease markers analysis in complex biological samples.     

Quantitative analysis of human serum leptin using a nanoarray protein chip based on single-molecule sandwich immunoassay

We report a method for the quantitative analysis of human serum leptin, which is a protein hormone associated with obesity, using a nanoarray protein chip based on a single-molecule sandwich immunoassay. The nanoarray patterning of a biotin-probe with a spot diameter of 150 nm on a self-assembled monolayer functionalized by MPTMS on a glass substrate was successfully accomplished using atomic force microscopy (AFM)-based dip-pen nanolithography (DPN). Unlabeled leptin protein molecules in human serum were detected based on the sandwich fluorescence immunoassay by total internal reflection fluorescence microscopy (TIRFM). The linear regression equation for leptin in the range of 100 zM-400 aM was determined to be y = 456.35x + 80,382 (R = 0.9901). The accuracy and sensitivity of the chip assay were clinically validated by comparing the leptin level in adult serum obtained by this method with those measured using the enzyme-linked immunosorbent assay (ELISA) performed with the same leptin standards and serum samples. In contrast to conventional ELISA techniques, the proposed chip methodology exhibited the advantages of ultra-sensitivity, a smaller sample volume and faster analysis time.     

Application of a polyaniline based ammonium sensor for the amperometric immunoassay of a urease conjugated Tal 1 protein

An amperometric immunoassay was developed by coupling the enzyme-linked immunosorbent assay (ELISA) microtiter-plate system with a polyaniline-perfluorosulfonated ionomer composite (PA/NF) electrode incorporated flow injection analysis (FIA) system and used for the analysis of Tal 1 protein, found in leukemic T cell. Rabbit polyclonal antibody (pAb) against Tal 1 and urease-pAb were used, respectively as the captured protein and enzyme labeled conjugate for sandwich immunoassay of Tal 1. Characteristics of the PA/NF electrode such as reproducibility, stability and sensitivity were studied. The detection limits of the PA/NF electrode for NH₄⁺ and urease were found to be 5 μM and 0.05 nM, respectively. Assay conditions such as the amount of pAb needed for coating the plate, the concentration of urease-pAb conjugate appropriate for the immunoreaction and the incubation time for urea to react with the bound urease-pAb in the microtiter-wells were also studied. A detection limit as lower as 0.5 ng/ml and a dynamic range of 1.0-100 ng/ml were found for the immunoassay of Tal 1 protein with the developed immunoassay system.     

Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) and its application for determination of amino acids from seeds and bryophyte plants using high-performance liquid chromatography with fluorescence detection and identification with electrospray ionization mass spectrometry

A pre-column derivatization method for the sensitive determination of amino acids and peptides using the tagging reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEOC. BCEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z (M + H)⁺ under electrospray ionization (ESI) positive-ion mode with an exception being Tyr detected at negative mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 246.2 corresponding to the cleavage of CO bond of BCEOC molecule. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3-4-fold molar reagent excess. Derivatives exhibit strong fluorescence and extracted derivatization solution with n-hexane/ethyl acetate (10:1, v/v) allows for the direct injection with no significant interference from the major fluorescent reagent degradation by-products, such as 1,2-benzo-3,4-dihydrocarbazole-9-ethanol (BDC-OH) (a major by-product), mono-1,2-benzo-3,4-dihydrocarbazole-9-ethyl carbonate (BCEOC-OH) and bis-(1,2-benzo-3,4-dihydrocarbazole-9-ethyl) carbonate (BCEOC)₂. In addition, the detection responses for BCEOC derivatives are compared to those obtained with previously synthesized 2-(9-carbazole)-ethyl chloroformate (CEOC) in our laboratory. The ratios AC_BCEOC/AC_CEOC = 2.05-6.51 for fluorescence responses are observed (here, AC is relative fluorescence response). Separation of the derivatized peptides and amino acids had been optimized on Hypersil BDS C₁₈ column. Detection limits were calculated from 1.0 pmol injection at a signal-to-noise ratio of 3, and were 6.3 (Lys)-177.6 (His) fmol. The mean interday accuracy ranged from 92 to 106% for fluorescence detection with mean %CV < 7.5. The mean interday precision for all standards was <10% of the expected concentration. Excellent linear responses were observed with coefficients of >0.9999. Good compositional data could be obtained from the analysis of derivatized protein hydrolysates containing as little as 50.5 ng of sample. Therefore, the facile BCEOC derivatization coupled with mass spectrometry allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids and peptides from biological and natural environmental samples.     

Automated polyvinylidene difluoride hollow fiber liquid-phase microextraction of flunitrazepam in plasma and urine samples for gas chromatography/tandem mass spectrometry

A new polyvinylidene difluoride (PVDF) hollow fiber (200 μm wall thickness, 1.2 mm internal diameter, 0.2 μm pore size) was compared with two other polypropylene (PP) hollow fibers (200, 300 μm wall thickness, 1.2 mm internal diameter, 0.2 μm pore size) in the automated hollow fiber liquid-phase microextraction (HF-LPME) of flunitrazepam (FLNZ) in biological samples. With higher porosity and better solvent compatibility, the PVDF hollow fiber showed advantages with faster extraction efficiency and operational accuracy. Parameters of the CTC autosampler program for HF-LPME in plasma and urine samples were carefully investigated to ensure accuracy and reproducibility. Several parameters influencing the efficiency of HF-LPME of FLNZ in plasma and urine samples were optimized, including type of porous hollow fiber, organic solvent, agitation rate, extraction time, salt concentration, organic modifier, and pH. Under optimal conditions, extraction recoveries of FLNZ in plasma and urine samples were 6.5% and 83.5%, respectively, corresponding to the enrichment factor of 13 in plasma matrix and 167 in urine matrix. Excellent sample clean-up was observed and good linearities (r² = 0.9979 for plasma sample and 0.9995 for urine sample) were obtained in the range of 0.1–1000 ng/mL (plasma sample) and 0.01–1000 ng/mL (urine sample). The limits of detection (S/N = 3) were 0.025 ng/mL in plasma matrix and 0.001 ng/mL in urine matrix by gas chromatography/mass spectrometry/mass spectrometry.     

Chemiluminescence detection of label-free C-reactive protein based on catalytic activity of gold nanoparticles

A novel, quantitative analytical method for measuring C-reactive protein (CRP) levels in human serum has been developed based on the catalytic activity of gold nanoparticles (GNPs) and luminol-H₂O₂ chemiluminescence (CL). The CL intensity in the presence of CRP and its ligand, O-phosphorylethanolamine (PEA), was greatly enhanced due to the aggregation of GNPs after the addition of 0.5 M NaCl. Any pretreatment steps, such as covalent functionalization of GNPs, addition of antibodies, or labeling of CRP, were not needed for CL detection. The CL enhancement was linearly proportional to CRP concentration in the range of 1.88 fM to 1.925 pM. The detection limit of CRP in serum samples was estimated to be as low as 1.88 fM. The detection sensitivity was increased more than 164 times of magnitude over that of the conventional, enzyme-linked immunosorbent assay (ELISA) method. This proposed GNP-based CL detection method offers the advantages of simplicity, rapidity, and sensitivity.     

A simple and sensitive LC-ESI-MS (ion trap) method for the determination of bupropion and its major metabolite, hydroxybupropion in rat plasma and brain microdialysates

A specific and highly sensitive liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) method for the direct determination of bupropion (BUP) and its main metabolite hydroxybupropion (HBUP) in rat plasma and brain microdialysate has been developed and validated. The analysis was performed on a Bonus RP C18 (100 mm × 2.1 mm i.d., 3.5 μm particles) column using gradient elution with the mobile phase consisting of acetonitrile and ammonium formate buffer (10 mM, pH 4). Plasma samples were analyzed after a simple, one-step protein precipitation clean-up with trichloroacetic acid (TCA), however clean-up for microdialysis samples was not necessary, enabling direct injection of the samples into the LC-ESI-MS system. Signals of the compounds were monitored under the multiple reaction monitoring (MRM) mode of the LC-ESI-MS (ion trap) for quantification. The precursor to product ion transitions of m/z 240-184 and m/z 256-238 were used to measure BUP and HBUP, respectively. The method was validated in both plasma and microdialysate samples, and the obtained lower limit of quantification (LLOQ) was 1.5 ng mL⁻¹ for BUP and HBUP in both matrices. The intra- and inter-day assay variability was less than 15% for both analytes. This LC-ESI-MS method provided simple sampling, rapid clean-up and short analysis time (<9 min), applicable to the routine therapeutic monitoring and pharmacokinetic studies of BUP and HBUP.     

A novel, environmentally friendly dispersive liquid-liquid microextraction procedure for the determination of copper

A novel, simple and environmentally friendly procedure for copper determination has been developed. The method is based on the formation of an ion associate of Cu(I) with 1,3,3-trimethyl-2-[5-(1,3,3-trimethyl-1,3-dihydroindol-2-ylidene)-penta-1,3-dienyl]-3H-indolium (DIDC) in the presence of chloride ions as ligand, followed by dispersive liquid-liquid microextraction (DLLME) of the formed ion associate into organic phase and UV-Vis spectrophotometric detection. The following experimental conditions were used: pH 3, 0.24 mol L^− 1 chloride ions, 0.06 mmol L^− 1 DIDC. The effect of the nature of the extraction solvent, auxiliary solvent and disperser solvent used was studied. A mixture of amyl acetate, tetrachloromethane, and methanol in a 1:1:3 v/v/v ratio was selected for the DLLME procedure. The absorbance of the coloured extracts at 640 nm wavelength obeys Beer's law in the range 0.020-0.090 mg L^− 1 of Cu. The limit of detection calculated from a blank test (n = 10) based on 3s is 0.005 mg L^− 1 of Cu. The developed procedure was applied to the analysis of water samples. The suggested DLLME is compared with two procedures previously reported from our laboratory based on (1) conventional liquid-liquid extraction, and (2) sequential injection extraction performed in a dual-valve sequential injection system. The advantages and disadvantages of each method are discussed.     

Improved methods for urinary atrazine mercapturate analysis--assessment of an enzyme-linked immunosorbent assay (ELISA) and a novel liquid chromatography-mass spectrometry (LC-MS) method utilizing online solid phase extraction (SPE)

Elimination of interfering substances in urine by solid phase extraction (SPE) prior to analysis resulted in 10-fold improvement in the sensitivity of atrazine mercapturate (AM) enzyme-linked immunosorbent assay (ELISA) compared to previous reports. Of the two tested SPE systems, Oasis^® HLB and MCX, the mixed-mode MCX gave good recoveries (82%) of AM in spiked samples measured by ELISA, whereas the reverse-phase HLB phase was not compatible with the immunochemical method. At relatively high concentrations of urinary AM (>20 ng mL⁻¹), sample dilution was effective enough for the elimination of interfering substances. The new liquid chromatography-mass spectrometry (LC-MS) method developed for AM utilizes online-SPE with Oasis^® HLB, column switching and a stable-isotope internal standard. The limit of quantification (0.05 ng mL⁻¹) indicates improved sensitivity compared with most previously published LC-MS methods for AM. Validation of all three methods, LC-MS, ELISA + SPE and ELISA + dilution with spiked urine samples showed good correlation between the known and measured concentrations with R² values of 0.996, 0.957 and 0.961, respectively. When a set (n = 70 plus 12 blind duplicates) of urine samples from farmers exposed to atrazine was analyzed, there was a good agreement (R² = 0.917) between the log normalized data obtained by ELISA + SPE and LC-MS. High correlation among the data obtained by the two tested methods and the LC-MS method by the Center of Disease Control and Prevention (CDC), together with low variability among the blind duplicates, suggests that both methods reported here would be suitable for the analysis of urinary AM as a biomarker for human exposure of atrazine.     

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) method for the measurement of 2,4-dichlorophenoxyacetic acid in human urine

An enzyme-linked immunosorbent assay (ELISA) method was developed to quantitatively measure 2,4-dichlorophenoxyacetic acid (2,4-D) in human urine. Samples were diluted (1:5) with phosphate-buffered saline containing 0.05% Tween and 0.02% sodium azide, with analysis by a 96-microwell plate immunoassay format. No clean up was required as dilution step minimized sample interferences. Fifty urine samples were received without identifiers from a subset of pesticide applicators and their spouses in an EPA pesticide exposure study (PES) and analyzed by the ELISA method and a conventional gas chromatography/mass spectrometry (GC/MS) procedure. For the GC/MS analysis, urine samples were extracted with acidic dichloromethane (DCM); methylated by diazomethane and fractionated by a Florisil solid phase extraction (SPE) column prior to GC/MS detection. The percent relative standard deviation (%R.S.D.) of the 96-microwell plate triplicate assays ranged from 1.2 to 22% for the urine samples. Day-to-day variation of the assay results was within ±20%. Quantitative recoveries (>70%) of 2,4-D were obtained for the spiked urine samples by the ELISA method. Quantitative recoveries (>80%) of 2,4-D were also obtained for these samples by the GC/MS procedure. The overall method precision of these samples was within ±20% for both the ELISA and GC/MS methods. The estimated quantification limit for 2,4-D in urine was 30 ng/mL by ELISA and 0.2 ng/mL by GC/MS. A higher quantification limit for the ELISA method is partly due to the requirement of a 1:5 dilution to remove the urine sample matrix effect. The GC/MS method can accommodate a 10:1 concentration factor (10 mL of urine converted into 1 mL organic solvent for analysis) but requires extraction, methylation and clean up on a solid phase column. The immunoassay and GC/MS data were highly correlated, with a correlation coefficient of 0.94 and a slope of 1.00. Favorable results between the two methods were achieved despite the vast differences in sample preparation. Results indicated that the ELISA method could be used as a high throughput, quantitative monitoring tool for human urine samples to identify individuals with exposure to 2,4-D above the typical background levels.     

Mixed hemimicelles SPE based on CTAB-coated Fe3O4/SiO2 NPs for the determination of herbal bioactive constituents from biological samples

In this paper, a solid-phase extraction (SPE) method based on mixed hemimicelles of cetyltrimethyl ammonium bromide (CTAB) on silica-coated magnetic nanoparticles (MNPs) is developed for extraction and preconcentration of compounds from the biological samples. We selected rhein and emodin which are the major active anthraquinones of rhubarb as model analytes. A high performance liquid chromatography-fluorescence detection (HPLC/FLD) method was developed for the determination of rhein and emodin in urine and serum samples. The main factors influencing the extraction efficiency including the amount of surfactant, the concentration of MNPs, the shaking time and the desorption ability of organic solvents were investigated and optimized. No interferences were caused by proteins or endogenous compounds in urine and serum samples. Good linearities (r² > 0.9995) for all calibration curves were obtained, and the limits of detection (LODs) for rhein and emodin were 0.2 and 0.5 ng/mL in urine samples and 7 and 10 ng/mL in serum samples, respectively. Satisfactory recoveries (92.76-109.90% and 97.53-107.72% for rhein and emodin) in the biological matrices were achieved.     

Determination of phenylurea herbicides in aqueous samples using partitioned dispersive liquid-liquid microextraction

Partitioned dispersive liquid-liquid microextraction (PDLLME), using THF as the dispersive solvent and dichloromethane as the extraction solvent, was utilized to isolate and concentrate phenylurea herbicides (PUHs) from aqueous samples. In PDLLME, a dispersive solvent should be able to partition in the organic extractant droplets to effectively extract the polar organic compounds from aqueous samples. The mixture of the water-immiscible extractant and the partitioned dispersive solvent was obtained by centrifugation, dried under low pressure, reconstituted in methanol-water mixture (1:1), and injected into a HPLC system for the determination of PUHs. The enrichment factors of the PUHs ranged from 68 to 126 under the optimal conditions. The linear range was 0.5-100 ng ml⁻¹ for each analyte, the relative standard deviations of PUHs were in the range of 1.5-5.9% (n = 5), and the detection limits (signal-to-noise ratio of 3) ranged from 0.10 to 0.28 ng ml⁻¹ for the herbicides. The range of intraday precision (n = 5) for PUHs at the levels of 0.5, 5, and 50 ng ml⁻¹ were 3.0-5.9%, 1.8-3.3%, and 2.2-3.6%, respectively. The range of interday precision (n = 5) at 0.5, 5, and 50 ng ml⁻¹ were 0.4-1.8%, 1.2-2.4%, and 0.9-2.3%, respectively. The recoveries of PUHs from three spiked river water samples, at a level of 10 ng ml⁻¹, were 91.2-104.1%. Due to its rapidity, ease of operation, and high recovery, PDLLME can be utilized to isolate and concentrate organic environmental contaminants such as PUHs from aqueous samples.     

A new high-speed hollow fiber based liquid phase microextraction method using volatile organic solvent for determination of aromatic amines in environmental water samples prior to high-performance liquid chromatography

A new and fast hollow fiber based liquid phase microextraction (HF-LPME) method using volatile organic solvents coupled with high-performance liquid chromatography (HPLC) was developed for determination of aromatic amines in the environmental water samples. Analytes including 3-nitroaniline, 3-chloroaniline and 4-bromoaniline were extracted from 6 mL basic aqueous sample solution (donor phase, NaOH 1 mol L⁻¹) into the thin film of organic solvent that surrounded and impregnated the pores of the polypropylene hollow fiber wall (toluene, 20 μL), then back-extracted into the 6 μL acidified aqueous solution (acceptor phase, HCl 0.5 mol L⁻¹) in the lumen of the two-end sealed hollow fiber. After the extraction, 5 μL of the acceptor phase was withdrawn into the syringe and injected directly into the HPLC system for the analysis. The parameters influencing the extraction efficiency including the kind of organic solvent and its volume, composition of donor and acceptor phases and the volume ratio between them, extraction time, stirring rate, salt addition and the effect of the analyte complexation with 18-crown-6 ether were investigated and optimized. Under the optimal conditions (donor phase: 6 mL of 1 mol L⁻¹ NaOH with 10% NaCl; organic phase: 20 μL of toluene; acceptor phase: 6 μL of 0.5 mol L⁻¹ HCl and 600 m mol L⁻¹ 18-crown-6 ether; pre-extraction and back-extraction times: 75 s and 10 min, respectively; stirring rate: 800 rpm), the obtained EFs were between 259 and 674, dynamic linear ranges were 0.1-1000 μg L⁻¹ (R > 0.9991), and also the limits of detection were in the range of 0.01-0.1 μg L⁻¹. The proposed procedure worked very well for real environmental water samples with microgram per liter level of the analytes, and good relative recoveries (91-102%) were obtained for the spiked sample solutions.