In situ derivatization/solid-phase microextraction: determination of polar aromatic amines

A solid-phase microextraction GC/MS method for the trace determination of a wide variety of polar aromatic amines in aqueous samples was developed. Prior to extraction the analytes were derivatized directly in the aqueous solution by diazotation and subsequent iodination in a one-pot reaction. The derivatives were extracted by direct-SPME using a PDMS/DVB fiber and analyzed by GC/MS in the full-scan mode. By diazotation/iodination, the polarity of the analytes was significantly decreased and as a consequence extraction yields were dramatically improved. The derivatization proved to be suitable for strongly deactivated aromatic amines and even the very polar diamino compounds can efficiently be enriched after derivatization. We investigated 18 anilines comprising a wide range of functional groups, which could be determined simultaneously. The method was thoroughly validated, and the precision at a concentration of 0.5 microg/L was 3.8-11% relative standard deviation for nonnitrated analytes using aniline-d(5) as internal standard and 3.7-10% for nitroaromatic amines without internal standard. The in situ derivatization/SPME/GC/MS method was calibrated over the whole analytical procedure and was linear over 2 orders of magnitude. Using 10-mL samples, detection limits of 2-13 ng/L were achieved for 15 of the 18 analytes. For two aminodinitrotoluene isomers and a diaminonitrotoluene, detection limits ranged from 27 to 38 ng/L. By allowing quantification at the 0.1 microg/L level, analysis of all target compounds meets EU drinking water regulations. The method provides high sensitivity, robustness, and high sample throughput by automation. Finally, the method was applied to various real water samples and in wastewater from a former ammunition plant the contents of several aromatic amines were quantified.     

Preconcentration and separation of nickel, copper and cobalt using solid phase extraction and their determination in some real samples

A solid phase extraction method has been developed to separate and concentrate trace amounts of nickel, cobalt and copper ions from aqueous samples for the measurement by flame atomic absorption spectrometry. By the passage of aqueous samples through activated carbon modified by dithioxamide (rubeanic acid) (DTO), Ni2+, Cu2+ and Co2+ ions adsorb quantitatively. The recoveries of analytes at pH 5.5 with 500 mg solid phase were greater than 95% without interference from alkaline, earth alkaline and some metal ions. The enrichment factor was 330. The detection limits by three sigma were 0.50 microg L(-1) for copper, 0.75 microg L(-1) for nickel and 0.80 microg L(-1) for cobalt. The loading capacity was 0.56 mg g(-1) for Ni2+, 0.50 mg g(-1) for Cu2+ and 0.47 mg g(-1) for Co2+. The presented procedure was applied to the determination of analytes in tap, river and sea waters, vegetable, soil and blood samples with successfully results (recoveries greater than 95%, R.S.D. lower than 2% for n=3).     

Development of a liquid chromatography-electrospray-tandem mass spectrometry method for the quantitative determination of benzoxazinone derivatives in plants

A new method for the quantification of benzoxazinone derivatives in extracts of wheat foliage and root samples using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) is described. Using this method, the characterization, separation, and quantitative detection of a mixture of six naturally occurring 1,4-benzoxazin-3(4H)-one derivatives, including the hydroxamic acids (DIMBOA, DIBOA), lactams (HBOA, and HMBOA), benzoxazilinones (BOA, MBOA), and two synthetic methoxylated variations of DIBOA and HBOA, was achieved. The application of a novel, highly modified reversed-phase LC column, the dodecyl (C12) TMS end-capped Synergi MAX-RP, enhanced the on-line chromatographic separation through improvements to component resolution, analyte stability and peak shape and also to the column lifetime. The complete ESI-MS-MS precursor-product ion fragmentation pathways for the benzoxazinone derivatives are described for the first time and used to deduce a generic fragmentation pattern for the compound class. Characteristic transitions for the benzoxazinones were thus used in the developed analytical method enabling reliable quantification with simultaneous screening for other potentially present derivatives, while eliminating interferences from other coeluting contaminants from the complex plant extract matrix. Quantitative analysis was done in the multiple reaction monitoring mode, using two specific combinations of a precursor-product ion transitions for each compound. The ESI-MS-MS detection method offered improvements to the sensitivity and selectivity, as compared with previously applied LC methods, with detection limits down to 0.002-0.023 ng/microL. The developed method was demonstrated by analyzing foliages and roots of six different wheat cultivars using pressurized liquid extraction-solid-phase extraction cleanup-LC-ESI-MS-MS. The analytes were detected in the range of 0.7-207 microg/g of dry weight.     

Analysis of pharmaceuticals in fish using liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method has been developed targeting 23 pharmaceuticals and 2 metabolites with differing physicochemical properties in fish tissue. Reversed-phase separation of target compounds was achieved using a C18 column and a nonlinear gradient consisting of 0.1% (v/v) formic acid and methanol. Eluted analytes were introduced into the mass analyzer using positive or negative electrospray ionization, as appropriate. A variety of extraction solvents, differing in polarity, pH, or both, were investigated in order to assess recovery of target compounds from 1-g tissue homogenates. Among 10 solvents tested, a 1:1 mixture of 0.1 M aqueous acetic acid (pH 4) and methanol was identified as optimal, resulting in extraction recoveries for 24 of 25 compounds exceeding 60%. Tissue extracts were found to influence the LC-MS/MS response for several analytes. Consequently, matrix-matched calibration standards were employed to determine analyte concentrations in environmental samples. Statistically derived method detection limits were <6 ng/g for most analytes. The method was subsequently used to screen for target analytes in fish from an effluent-dominated stream. Diphenhydramine, diltiazem, carbamazepine, and norfluoxetine were detected in 11 of 11 environmental samples at concentrations ranging from 0.11 to 5.14 ng/g.     

Quantifying phthalate metabolites in human meconium and semen using automated off-line solid-phase extraction coupled with on-line SPE and isotope-dilution high-performance liquid chromatography--tandem mass spectrometry

We developed an analytical method using off-line solid-phase extraction (SPE) coupled with on-line SPE and isotope-dilution high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to determine the concentrations of phthalate metabolites in human meconium and in semen. First, we used off-line SPE to remove interfering proteins and other biomolecules from the samples. Then, we preconcentrated the phthalate metabolites in the extract using on-line SPE before measuring them by HPLC-MS/MS. For most of the analytes, the limits of detection ranged between 0.2 and 0.7 ng/g for meconium and between 0.3 and 0.7 ng/mL for semen. The recovery after off-line SPE varied for most analytes between 65 and 99% at concentrations ranging from 3.0 to 30.0 ng/mL in semen and between 67 and 103% at concentrations ranging from 2.0 to 10.0 ng/mL in meconium. Precision measured by the relative standard deviation ranged from 3.2 to 19.1% for intraday and from 3.9 to 18.6% for interday. We validated this novel approach--which is applicable to other biological matrixes, including serum and breast milk--on spiked samples and on five meconium samples and one pooled semen sample from people with no known occupational exposure to phthalates.     

Simultaneous analysis of multiple classes of antibiotics by ion trap LC/MS/MS for assessing surface water and groundwater contamination

Solid-phase extraction (SPE) and liquid chromatography in combination with ion trap mass spectrometry (LC/MS/MS) conditions were optimized for the simultaneous analysis of 13 antibiotics belonging to multiple classes and caffeine in 3 different water matrixes. The single-cartridge extraction step was developed using a reversed-phase cartridge, resulting in recoveries for the 14 compounds ranging from 71 to 119% with relative standard deviations of 16% or lower. The analytes were separated in one chromatographic run, and the SPE-LC/MS/MS detection limits ranged from 0.03 to 0.19 microg/L. The SPE procedure was validated in groundwater, surface water, and wastewater. The analysis of samples from each of the three water matrixes revealed clindamycin (1.1 microg/L) in surface water and multiple antibiotics in wastewater (0.10-1.3 microg/L). The use of identification points to unambiguously assign the identity of antibiotics in various water matrixes was applied to an ion trap data-dependent scanning method, which simultaneously collects full scan and full scan MS/MS data for the unequivocal identification of target analytes.     

Derivatization for LC-electrospray ionization-MS: a tool for improving reversed-phase separation and ESI responses of bases, ribosides, and intact nucleotides

We have developed a method for analyzing polar compounds by reversed-phase LC-ESI-MS following esterification of the analytes' free hydroxyl groups with propionyl or benzoyl acid anhydride. The method was applied to members of the plant hormone group cytokinins, which includes adenine bases, ribosides/glycosides, and nucleotides substituted at N-6 with an isoprenoid side chain, spanning a wide range of polarity. It was also used to analyze other compounds of biological importance, e.g., the nucleotides AMP, ADP, and ATP. The formation of more hydrophobic derivatives had a significant impact on two aspects of the analysis. The retention on a reversed-phase material was greatly increased without the use of any acetate/formate buffer or ion pairing reagent, and the ESI response was enhanced, due to the higher surface activities of the derivatives. Detection limits of propionylated cytokinins were in the high-attomole to low-femtomole range, an improvement by factors of 10-100 compared to previously reported figures. Using an automated SPE-based purification method, 12 endogenous cytokinins were quantified in extracts from 20- to 100-mg samples of leaves (from the plant Arabidopsis thaliana) with high accuracy and precision. Furthermore, the chromatographic properties of the benzoylated AMP, ADP, and ATP in the reversed-phase LC-MS system were much better in terms of retention, separation, and sensitivity than those of their underivatized counterparts, even without the use of any ion pairing reagent. Our data show that derivatization followed by LC-ESI-MS is an effective strategy for analyzing low molecular weight compounds, enabling compounds with a wide range of polarity to be determined in a single-injection LC-MS analysis.     

Pressurized hot water extraction coupled on-line with LC-GC: determination of polyaromatic hydrocarbons in sediment

Pressurized hot water extraction (PHWE) was directly combined with a LC-GC system for the determination of polyaromatic hydrocarbons (PAHs) in sediment. The sediment sample was first extracted with pressurized hot water, and the analytes were adsorbed into a solid-phase trap. The trap also functioned as a LC column, which removed most of the interfering matrix components. The 780-microL LC fraction containing the analytes was directly transferred to the GC using an on-column interface. The whole PHWE-LC-GC analysis took place in a closed system, and no sample pretreatment was required. The sensitivity of the method was excellent due to the efficient concentration in the LC-GC system. Sensitivity was approximately 800 times better than in traditional systems. In addition, only a small amount of sample (10 mg) was required for the analysis. The PHWE-LC-GC method proved to be linear in the concentration range of 0.01-2 microg/g, the limits of quantification were below 0.01 microg/g for all the analytes, and the relative standard deviations were between 3 and 28%. LC cleanup and the improved sensitivity made detection with FID sufficient for the determination of analytes. The results were comparable to those obtained in an interlaboratory comparison study as well as to the results obtained with off-line SFE-GC-MS.     

Determination of carbamazepine and its metabolites in aqueous samples using liquid chromatography-electrospray tandem mass spectrometry

A quantitative method is described for solid-phase extraction (SPE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous analysis of carbamazepine and its five metabolites, 10,11-dihydro-10,11-epoxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine, 2-hydroxycarbamazepine, 3-hydroxycarbamazepine, and 10,11-dihydro-10-hydroxycarbamazepine. An SPE procedure was used to concentrate target compounds from aqueous samples collected from sewage treatment plant (STP) wastewater and surface water. Extracts were analyzed using electrospray LC-MS/MS with time-scheduled selected reaction monitoring. The recoveries of the analytes were 83.6-102.2% from untreated sewage (influent), 90.6-103.5% from treated sewage (effluent), and 95.7-102.9% from surface water samples. The instrumental detection limits were 0.8-4.8 pg for the analytes. Matrix effects were investigated for the analytes in HPLC-grade water, surface water, and STP influent and effluent. Ion suppression increased for analytes in order of surface water to STP effluent to STP influent, but no ion suppression was observed for analytes in HPLC-grade water. The developed method was validated by analysis of environmental aqueous samples: STP influent and effluent and surface water. Carbamazepine and all five metabolites were detected in STP influent and effluent samples. Only carbamazepine and 10,11-dihydro-10,11-dihydroxycarbamazepine were detected in the surface water sample. Notably, 10,11-dihydro-10,11-dihydroxycarbamazepine was detected at approximately 3 times higher concentrations than the parent drug, carbamazepine, in all of the aqueous samples. To our knowledge, this is the first report on the simultaneous determination of carbamazepine and its metabolites in environmental samples.     

Development of cloud point extraction for simultaneous extraction and determination of gold and palladium using ICP-OES

Cloud point method was applied for the simultaneous extraction and preconcentration of trace amounts of gold and palladium. The extraction of analytes was performed in the presence of 1,8-diamino-4,5-dihydroxy anthraquinone as chelating agent and Triton X-114 as a non-ionic surfactant. After phase separation, the surfactant-rich phase was diluted with concentrated HNO(3) (65%, w/w) and the analytes concentrations were determined by inductively coupled plasma-optical emission spectrometry (ICP-OES). The variables affecting the complexation and extraction conditions were optimized and under the optimum conditions (i.e. pH 6.5, 2.2 x 10(-4) mol l(-1) chelating agent, 0.15% (w/v) of Triton X-114, equilibration temperature 55 degrees C, centrifuge at 3500 rpm), quantitative extraction of Au(III) and Pd(II) from 100 ml of the aqueous solution was performed. The calibration curves were linear in the range of 0.5-1000 microg l(-1) with detection limits of 0.5 and 0.3 microg l(-1) and the enrichment factors were 8.6 and 20.2 for Au and Pd, respectively. Also the precision (%RSD) for eight replicate determinations of the analytes was better than 5%. Finally, the proposed method was successfully applied for the determination of Au and Pd in mine stones and standard reference materials (SRM).     

Three modified activated carbons by different ligands for the solid phase extraction of copper and lead

In the presented work, 5,5-diphenylimidazolidine-2,4-dione (phenytoin) (DFTD), 5,5-diphenylimidazolidine-2-thione-,4-one (thiophenytoin) (DFID) and 2-(4'-methoxy-benzylidenimine) thiophenole (MBIP) modified activated carbons have been used for the solid phase extraction of copper and lead ions prior to their flame atomic absorption spectrometric determinations. The influences of the various analytical parameters including pH, amounts of reagent, sample volume and eluent type, etc. on the recovery efficiencies of copper and lead ions were investigated. The influences of alkaline, earth alkaline and some transition metals on the adsorption of the analytes were also examined. The detection limits by three sigma for analyte ions were 0.65 and 0.42 microg L(-1) using activated carbon modified with DFID; 0.52 and 0.37 microg L(-1) using activated carbon modified with DFTD and 0.46 and 0.31 microg L(-1) using activated carbon modified with MBIP for Pb(II) and Cu(II), respectively. The procedure was applied to the determination of analytes in natural waters, soil, and blood samples with satisfactory results (recoveries greater than 95%, R.S.D.'s lower than 4%).     

LC-MS/MS approach for quantification of therapeutic proteins in plasma using a protein internal standard and 2D-solid-phase extraction cleanup

The bioanalysis of plasma samples generated from in vivo studies of therapeutic proteins is of increasing interesting in the biopharmaceutical industry. The conventional ELISA approach has a long assay development time which can limit use in the early discovery and development of protein-based drugs. In this study, an LC-MS/MS bioassay was developed for the quantification of somatropin and a therapeutic human monoclonal antibody. The assay used bovine fetuin as an internal standard and a two-dimensional solid-phase extraction for the cleanup of the plasma digest. Sample extracts were resolved on an analytical size column using a 6 min LC gradient and analyzed using a triple-quadruple mass spectrometer. The linearity of the assay for somatropin was established from 1 to 1000 microg/mL with accuracy and precision within 15%. This LC-MS approach was also applied to a rat pharmacokinetic study of the therapeutic monoclonal antibody with a lower quantitation limit of 0.5 microg/mL. The LC-MS assay had improved accuracy and precision, and the results from analysis of in vivo study samples showed good agreement with the data obtained with an ELISA. The results from this study indicate that the LC-MS bioassay is a simple and feasible approach for the bioanalysis of therapeutic proteins to support in vivo studies during early drug discovery and development.     

Neurosteroids in rat brain: extraction, isolation, and analysis by nanoscale liquid chromatography-electrospray mass spectrometry

A method designed for the analysis of sulfated neurosteroids and unconjugated ketonic neurosteroids in rat brain using nanoscale liquid chromatography-electrospray (nano-LC-ES) mass spectrometry is described. Neurosteroids in rat brain tissue were extracted, purified, and separated into two groups, neutral unconjugated steroids and steroid sulfates, by employing solid-phase partition, cation- and anion-exchange chromatography. The steroid sulfate fraction was analyzed by nano-LC-ES mass spectrometry. Contrary to expectations, the sulfates of pregnenolone and dehydroepiandrosterone (DHEA) were not detected. Internal standards, including pregnenolone sulfate, were recovered and the detection limit of the method was 0.3 ng/g of wet brain. Cholesterol sulfate was detected at a level of 1.2 microg/g of wet brain. The neutral unconjugated steroid fraction was derivatized with hydroxylamine hydrochloride to convert oxosteroids into their oximes. The oximes were isolated using cation-exchange chromatography and were analyzed by nano-LC-ES tandem mass spectrometry. The analyses of the neutral unconjugated steroid fraction confirmed the presence in rat brain of pregnenolone, pregnanolone isomers, progesterone, testosterone, and DHEA, which were characterized by their retention times, the mass of the protonated molecules, and characteristic fragment ions. The levels were estimated by addition of [3,4-(13)C(2)]-progesterone as an internal standard and found to be in a range of 0.04-20 ng/g.     

Confirmation and quantification of hemoglobin-based oxygen carriers in equine and human plasma by hyphenated liquid chromatography tandem mass spectrometry

Oxyglobin (OXY) and Hemopure (HMP) are produced from bovine hemoglobin (Hb) and were developed for the treatment of anemia in animal and human patients, respectively. Hemolink (HML) is a blood substitute of human Hb origin under development. The ability of these agents to carry oxygen in circulating blood and their promise to improve oxygen delivery to tissues supports the potential for their abuse in equine and human athletes. To deter athletes from abuse of these agents, a method has been developed for the detection, confirmation and quantification of OXY, HMP, and HML in equine and human plasma. OXY, HMP, and HML were extracted from equine or human plasma by solid-phase extraction using Bond Elut ENV cartridges and were digested by trypsin at 37 degrees C for 3 h. The tryptic digests were analyzed by LC-MS/MS, and tryptic peptides specific for bovine and human Hbs were targeted. OXY and HMP were detected, quantified, and confirmed using the y14 ion and b8 ion of the tryptic peptide from bovine Hb alpha chain residues 69-90, and HML was quantified using the tryptic peptide from human Hb alpha chain residues 63-91. The limit of detection for OXY in equine plasma and HML in human and equine plasma was 50 and 250 microg/mL for HMP in human and equine plasma. The limit of confirmation was 250 microg/mL for OXY in equine plasma, 500 microg/mL for HML in human and equine plasma, and 1000 microg/mL for HMP in human and equine plasma. The linear range for quantification was 50-5000 microg/mL for OXY in equine plasma and for HML in human and equine plasma, and 250-5000 microg/mL for HMP in human and equine plasma. The intraday and interday CV were less than 17% for quantification of OXY in equine plasma with external calibration. OXY was stable for more than 30 days at -20 and -70 degrees C. OXY was detected and quantified in equine plasma up to 24 h following administration of a very low dose of OXY (32.5 g in 2 x 125 mL per horse), and its presence in equine plasma was confirmed up to 12 h postadministration.     

A liquid chromatography-mass spectrometry assay for analyzing sulfonamide antibacterials in cattle and fish muscle tissues

A simple and rapid method able to determine residues of 12 sulfonamide (SAs) antibacterials in cattle and trout muscle tissues is presented. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography-mass spectrometry (LC-MS). The LC-MS instrumentation was equipped with an electrospray source and a single quadrupole. After 0.8 g of a flesh sample containing the analytes is deposited on sand (crystobalite), this material is packed into an extraction cell. SAs are extracted by flowing 4 mL of water through the cell heated at 80 degrees C. A 0.5-mL aliquot of the bovine tissue extract is then directly injected into the LC column, while the fish tissue extract is filtered prior to LC-MS analysis. MS data acquisition was performed in the positive-ion mode and monitoring at least three ions for each target compound. Confirmatory ions were produced by the in-source collision-induced dissociation process. At the tolerance levels issued by the EU and U.S. Food and Drug Administration, i.e., 100 ppb, recovery of the analytes in bovine and trout muscle tissues was 75-98% with RSDs ranging between 1 and 8%. Estimated limits of quantification (S/N = 10) were 6-15 ppb for SAs in bovine muscle tissue and 3-13 ppb in trout fillet. When trying to reduce the analysis time by using a short chromatographic run time, severe ion signal suppression was experienced for the early-eluted SAs. This effect was traced to competition effects by polar endogenous coextractives, maybe proteinaceous species, which are eluted in the first part of the chromatographic run. This unwelcome effect was removed by simply adopting more selective chromatographic conditions.     

Simultaneous determination of halogenated derivatives of alkylphenol ethoxylates and their metabolites in sludges, river sediments, and surface, drinking, and wastewaters by liquid chromatography-mass spectrometry

A quantitative solid-phase extraction-liquid chromatography/mass spectrometry (SPE-LC/MS) method is described for the simultaneous analysis of halogenated byproducts of alkylphenolic compounds and their degradation products formed during chlorine disinfection in the presence of bromide ions. Compounds analyzed include brominated and chlorinated nonylphenol ethoxylates (XN-PEOs); octylphenol ethoxylates (XOPEOs); nonylphenols (XNP); nonylphenoxycarboxylates (XNPECs) and their precursors nonionic surfactants, alkylphenol ethoxylates (APEOs); and their metabolites formed during sewage treatment, alkylphenoxycarboxylates (APECs) and alkylphenols (APs). Target compounds were concentrated from water samples using a C18 SPE procedure. Extracts were analyzed using reversed phase LC/MS. The performances of both atmospheric pressure chemical ionization (APCI) and electrospray (ESI) interfaces were compared. ESI offered better sensitivity and specificity for a higher range of oligomers. Detection limits (LODs) for water samples were from 20 to 100 ng/L; and for sediment samples, from 2 to 10 microg/kg. Slightly higher LODs were obtained for sludge samples (5-25 microg/kg). Halogenated byproducts were found in sludge from Barcelona drinking water treatment plant in concentrations of 220 microg/kg for BrNP, 430 microg/kg for BrNPEOs (nEO = 1 - 2), and 1600 microg/kg for BrNPEOs (nEO = 3 - 15). The concentration of ClNPEOs was estimated to be in the order of 660 microg/kg (assuming the same response as BrNPEOs). Halogenated OPEOs were also identified, and their concentration was approximately 50 times lower than the concentration of NPEOs analogues. To our knowledge, this is the first method described that allows simultaneous determination of alkyphenol ethoxylates and halogenated derivatives, including degradation products.     

Ion-pair RP-HPLC determination of sugars, amino sugars, and uronic acids after derivatization with p-aminobenzoic acid

A new, selective, and sensitive ion-pair RP-HPLC method for the simultaneous determination of three classes of natural organic compounds, i.e., carbohydrates, amino sugars, and uronic acids, in environmental samples is presented. p-Aminobenzoic acid is used for precolumn derivatization of the analytes, enabling fluorescence (lambda(ex) 313 nm, lambda(em) 358 nm) or photometric detection (303 nm). The dependence of the derivatization yield on the reaction conditions is examined. Derivatives of lactose, galactose, glucose, mannose, xylose, arabinose, galacturonic acid, glucuronic acid, N-acetylglucosamine, and glycerinealdehyde were separated on a RP-C18 column with hydrophilic end capping within 35 min, applying TBAHSO4 as the ion-pair reagent. The concentration detection limits range between 20 and 30 microg L(-1) ((1-2) x 10(-7) M) for fluorescence detection and between 30 and 75 microg L(-1) for UV detection. A good linearity is achieved in the concentration range from 50 microg L(-1) to 100 mg L(-1) (r2 > 0.99). The described method has been applied for the determination of mono-/disaccharides, uronic acids, and amino sugars in soil solutions and in landfill leachates.     

A single calibration graph for the direct determination of ascorbic and dehydroascorbic acids by electrogenerated luminescence based on Ru(bpy)(3)2+ in aqueous solution

Ascorbic (H2A) and dehydroascorbic (DA) acids were for the first time directly determined in a single chromatographic run by means of the tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)(3)2+) based electrogenerated chemiluminescence (ECL) detection. For the first time, it was demonstrated that DA, a nonelectroactive compound, is ECL active and is responsible for the ECL behavior of H2A. This fact, together with the lack of a DA standard, suggested the use of a calibration graph obtained for H2A, for determining both analytes. The proven ECL activity of DA, together with literature data relative to the standard redox potentials of the different species coming from H2A, led to a reconsideration of the proposed ECL reaction mechanism for H2A. The role of the OH- ion in the reaction mechanism of the two analytes appeared to be crucial. H2A and DA could be separated by a suitable C18-reversed-phase HPLC column using an aqueous 30 mM H3PO4 solution as the mobile phase. The optimal ECL response was achieved by polarizing the working electrode at 1.150 Vvs SCE (standard calomel electrode) (oxidation diffusion limiting potential for both H2A and Ru(bpy)(3)2+). The Ru(bpy)(3)2+ solution, at pH 10 for carbonate buffer, was mixed to the eluent solution in a postcolumn system, obtaining, still at pH 10, the final 0.25 mM Ru(bpy)(3)2+ concentration. The detection limit found for the two analytes was 1 x 10(-7) M. The method was successfully applied to the determination of the analytes in a commercially available orange fruit juice.     

Fully automated 96-well liquid-liquid extraction for analysis of biological samples by liquid chromatography with tandem mass spectrometry

A fully automated high-throughput liquid-liquid extraction (LLE) methodology has been developed for preparation of biological samples using a 96-well LLE plate and a 96-channel robotic liquid handling workstation. The 96-well LLE plate is made of a 96-well filter plate filled with inert diatomaceous earth particles, allowing continuous and efficient extraction of analytes between the aqueous biological sample and the organic extraction solvent. Two carboxylic acid-based protease inhibitor compounds with high and low levels of plasma protein binding were chosen for the development and application of the automated methodology. The LLE extracts of the plasma samples of the two compounds were analyzed by high-performance liquid chromatography with electrospray (ESI) tandem mass spectrometry (LC-MS/MS). The LC-MS/MS method was developed using a rapid gradient LC separation, followed by sample introduction through an ionspray interface in the negative ion mode and tandem mass spectrometric detection with selected reaction monitoring. In the optimized LLE method, a formate buffer solution was first loaded into a 96-well filter plate packed with inert diatomaceous earth material. Then crude plasma samples and a water-immiscible organic solvent, methyl ethyl ketone, were sequentially added to the LLE plate so that LLE would occur in the interface between the two liquid phases on the surface of individual particles in each well. The organic eluate containing extracted analytes was evaporated and reconstituted for LC-MS/MS analysis. This fully automated LLE methodology avoids several disjointed steps involved in a manual or semiautomated LLE method, leading to significantly reduced sample preparation time, increased sample throughput, and clean sample extracts for improved ESI-MS/MS detection. The automated LLE methodology is universal and can be employed for sample preparation of other biological fluids. The complete bioanalytical method, based on the automated LLE and fast gradient LC-MS/MS, was validated and successfully applied to the quantitative analysis of protease inhibitors in rat plasma.     

A biosorption system for metal ions on Penicillium italicum-loaded on Sepabeads SP 70 prior to flame atomic absorption spectrometric determinations

A solid phase extraction (SPE) preconcentration system, coupled to a flame atomic absorption spectrometer (FAAS), was developed for the determination of copper(II), cadmium(II), lead(II), manganese(II), iron(III), nickel(II) and cobalt(II) ions at the microg L(-1) levels on Penicillium italicum-loaded on Sepabeads SP 70. The analytes were adsorbed on biosorbent at the pH range of 8.5-9.5. The adsorbed metals were eluted with 1 mol L(-1) HCl. The influences of the various analytical parameters including pH of the aqueous solutions, sample volume, flow rates were investigated for the retentions of the analyte ions. The recovery values are ranged from 95-102%. The influences of alkaline, earth alkaline and some transition metal ions were also discussed. Under the optimized conditions, the detection limits (3s, n=21) for analytes were in the range of 0.41microg L(-1) (cadmium) and 1.60microg L(-1) (iron). The standard reference materials (IAEA 336 Lichen, NIST SRM 1573a Tomato leaves) were analyzed to verify the proposed method. The method was successfully applied for the determinations of analytes in natural water, cultivated mushroom, lichen (Bryum capilare Hedw), moss (Homalothecium sericeum) and refined table salt samples.