Teaching social responsibility in analytical chemistry.

Analytical chemistry is key to the functioning of a modern society. From early days, ethics in measurements have been a concern and that remains today, especially as we have come to rely more on the application of analytical science in many aspects of our lives. The main aim of this Feature is to suggest ways of introducing the topic of social responsibility and its relation to analytical chemistry in undergraduate or graduate chemistry courses.

Cost-effective flow injection spectrophotometric assay of iron content in pharmaceutical preparations using salicylate reagent.

A new flow injection procedure for an assay of Fe(III) by using salicylate obtained from antipyretic powder, which is a cheap and easily available reagent, is proposed. A red complex was continuously monitored by a laboratory-made green LED colorimeter. A linear calibration was obtained in the range of 1-20mgFel(-1) with a detection limit of 0.5mgFel(-1) and R.S.D.s of 1.4-5.4% (n=3, for 1-20mgFel(-1)). The new procedure was applied to assay iron contents in pharmaceutical preparations. The results were in good agreement with those of the USP standard method.

Stopped-flow injection simultaneous determination of phosphate and silicate using molybdenum blue.

Kinetic information for the phosphate-molybdate-ascorbic acid reaction can be obtained by making use of a very simple manually operated stopped-flow injection (FI) system. Various parameters (concentrations of reagents, flow rate, mixing coils, and volume of flow cell) were investigated for determination of phosphate. A stopped-FI system should be arranged for low degree of mixing (of reactants) and low dispersion so that good signals of rate changes will be observed. Simultaneous determination of phosphate and silicate by the stopped-FI technique is proposed, using a laboratory-made semi-automatic stopped-FI Analyzer with LED-based photometer. It is based on kinetic separation of phosphate and silicate using molybdenum blue. The proposed procedure has been demonstrated for the application to water samples. The results obtained agree with that of a standard method.

Determination of trace iron in beer using flow injection systems with in-valve column and bead injection.

Three flow injection (FI) systems were investigated for the determination of trace iron in beer: an FI-in-valve column-flame atomic absorption spectrophotometry (FI-FAAS) system, a spectrophotometric FI system with a column placed at the detection point, and an FI-spectrophotometric system with bead injection (FI-BI). Cationic exchange resin Dowex 50W X8 and iminodiacetate chelating resin, Chelex-100, were employed for the FI-spectrophotometric and FI-FAAS systems, respectively. The FI-in-valve column, packed with the resin, enhances the FAAS performance. The spectrophotometric FI system with a column (packed with Chelex-100) placed at the detection point (in a cell holder of a spectrophotometer) is based on the formation of iron (II)-1,10-phenanthroline complex sorbed onto the resin. No eluent has been found to be suitable. The FI-BI for renewable microcolumn has been proven to be an alternative. The FI-FAAS and FI-BI procedures provide online sample preseparation and preconcentration for the determination of iron in beer. Both are simple, rapid, and economical. The procedures also involve sample preparation (decarbonation and suppression of tannin interference by adding ascorbic acid) and standard addition. The results obtained by FI-FAAS and FI-BI agree with those of AOAC spectrophotometric method.

Postmortem forensic toxicology of selective serotonin reuptake inhibitors: a review of pharmacology and report of 168 cases.

This paper reviews the complex pharmacology of the new class of antidepressant medications exhibiting selective inhibition of serotonin reuptake. The four selective serotonin reuptake inhibitors (SSRIs) considered--fluoxetine, fluvoxamine, sertraline and paroxetine--can result in toxicity and death through contributing to serotonergic excess resulting in serotonin syndrome, inhibiting the metabolism of other centrally acting drugs, leading to accumulation of toxic concentrations, and exerting complex vasoactive effects on the vascular smooth muscle. This latter feature is of particular concern in patients with preexisting heart disease. An analytical method involving isolation of the drugs by liquid/liquid extraction at alkaline pH into n-butyl chloride, and analysis by gas chromatography/mass spectrometry (GC/MS) is described, together with some of its limitations. Toxicologic and cause and manner of death data were examined in 60 deaths involving fluoxetine, 5 involving fluvoxamine, 75 involving sertraline, and 28 involving paroxetine. Deaths involving drug toxicity were generally a result of ingestion of multiple drugs, and in only a small number of the cases was death attributed principally to the SSRI involved. The potential for drug interactions between members of this class of drugs is discussed as well as their metabolites and a variety of other therapeutic and abused drugs which can contribute to their toxicity. In the absence of other risk factors, the lowest concentrations determined to have resulted in death were 0.63 mg/L for fluoxetine, 0.4 mg/L for paroxetine, and 1.5 mg/L for sertraline. We had insufficient data to make even this crude assessment for fluvoxamine. Drug-induced elevation of serotonin concentrations may be a significant risk factor for patients with atherosclerotic cardiovascular disease (ASCVD). Other factors including preexisting disease and the presence of other drugs and their pharmacology need to be carefully considered before determining the appropriate cause and manner of death in these cases.

Measurement of cellular stimulation through monitoring pH changes by bead injection fluorescence microscopy.

This work presents a method for extracellular and intracellular pH measurements in live cells based on a combination of the bead injection (BI) technique and fluorescence microscopy. For extracellular pH measurement, cells are grown on fluorescent beads, packed into a small column by a sequential injection instrument, and fluorescence intensity from the beads stained by the indicator is recorded by a fluorescence microscope. The method is applied to quantifying carbachol stimulation of Chinese hamster ovary (CHO) cells transfected with the m1 muscarinic receptor and is verified by a glucose depletion experiment. The results yield an EC(50) value of 1 muM for carbachol, which is in reasonable agreement with the literature value 3 muM determined by an existing potentiometric technique for measuring acid release. The intracellular measurement utilizes CHO M1 cells growing on non-fluorescent beads. For this method the cells rather than the beads are stained by incubating them in a solution of the fluorescent pH indicator BCECF. The cells are also stimulated with carbachol and the intracellular pH dependent fluorescence from the cells is recorded. The results show dependence between intracellular pH changes and carbachol concentration and yield an EC(50) value of 4 muM.

Experimental study of kinematic focusing. Comparison of electroinjection and sequential injection determination of copper.

The recently predicted phenomenon of kinematic focusing was studied experimentally using copper ions and EDTA as reactants. Kinematic focusing occurs, in electroinjection analysis, when the detected reaction product moves at the same rate as the reagent present in excess. Thus, reaction product accumulates without dispersion at the front of the excess reagent. Cu-EDTA(2-) complex was observed at 254 nm to form an exceptionally sharp peak as the front of the EDTA zone passed by the detector. The concentrating effect of kinematic focusing was quantified by electroinjection of premixed Cu-EDTA(2-). Sensitivity was compared to that of sequential injection analysis using a 1 cm optical pathlength. Sensitivity was highest in the electroinjection mode, in spite of its 120 mum capillary pathlength, due to kinematic focusing.

Hydrodynamics and mass transfer of the coaxial jet mixer in flow injection analysis.

A coaxial jet mixer that was previously proposed for rapid and efficient mixing under laminar flow conditions has been studied both theoretically and experimentally. A mathematical model that consists of a set of Navie-Stokes equations that determine the flow velocities and three diffusion-convection reaction equations that determine the reactant and product concentrations has been developed. Equations are solved with the help of finite difference techniques for different flow conditions. The quality of sample and reagent mixing is characterized by the mean product concentration and the amount of product produced. Theoretical results are compared with experimental ones for the mixing of bromothymol blue (a pH indicator) in the outer capillary with NaOH in the inner capillary of the jet mixer.

Identification of tramadol and its metabolites in blood from drug-related deaths and drug-impaired drivers.

Tramadol is a centrally acting, binary analgesic that is neither an opiate-derived nor a nonsteroidal anti-inflammatory drug and that was approved for use in the United States in 1995. It is used to control moderate pain in chronic pain settings such as osteoarthritis and postoperative cases. Used in therapy as a racemic mixture, the (+)-enantiomer weakly binds to the mu-opioid receptor, and both enantiomers inhibit serotonin and norepinephrine reuptake. Tramadol's major active metabolite, O-desmethyltramadol (ODT), shows higher affinity for the mu-opioid receptor and has twice the analgesic potency of the parent drug. The synergism of these effects contributes to tramadol's analgesic properties with the (+)-enantiomer exhibiting 10-fold higher analgesic activity than the (-)-enantiomer. Although tramadol was initially thought to exhibit low abuse potential, Ortho-McNeil, the drug's manufacturer, recently reported a large number of adverse events attributed to tramadol including abuse by opioid-dependent patients, allergic reactions, and seizures. The high number of adverse reactions has prompted the company to update the prescribing information for the drug. An analytical method using gas chromatography-mass spectrometry (GC-MS) without derivatization for the determination of tramadol and its metabolites is reported. An n-butyl chloride extraction is followed by GC-MS analysis using a 5% phenylmethylsilicone column (30 m x 0.32-micron i.d.). Analysis of 12 blood samples from tramadol-related deaths and four nonfatal intoxications involving tramadol revealed concentrations ranging from 0.03 to 22.59 mg/L for tramadol, from 0.02 to 1.84 mg/L for ODT, and from 0.01 to 2.08 mg/L for N-desmethyltramadol. Three deaths were clearly attributable to acute morphine toxicity, one was a doxepin overdose, and six were multiple drug overdoses. The role of tramadol in each death is explored.

Flow injection renewable surface immunoassay for real time monitoring of biospecific interactions.

An automated system for performing rapid immunoassay, kinetic measurements, and affinity ranking of biomolecular interactions using fluorescence-labeled ligands is described. Its distinctive feature is the automated renewal of solid phase for each measurement, which avoids the need for regeneration of the sensing surface. This system--flow injection renewable surface immunoassay (FIRSI)--is used for the first time here for determination of rate constants for an antibody/antigen interaction and for affinity ranking of several related antigens against one antibody. The performance of the system is compared with a commercial BIAcore system that uses surface plasmon resonance for monitoring biomolecular interactions. While the values of association and dissociation rate constants for human serum albumin (HSA) with monoclonal anti-HSA antibody obtained by these techniques were comparable, it is shown that the FIRSI techniques requires simpler instrumentation, handles a broader size range of analytes, and does not suffer from disturbances caused by changes in the refractive index.

Sequential injection wetting film extraction applied to the spectrophotometric determination of chromium(VI) and chromium(III) in water.

The spectrophotometric determination of Cr(VI) and Cr(III) via sequential injection was used to demonstrate the sensitivity enhancement provided by a newly developed wetting film extraction system. The reaction product of Cr(VI) with 1,5-diphenylcarbazide was ion-paired with perchlorate and extracted into an organic wetting film consisting of octanol and 4-methyl-2-pentanone on the inner wall of a Teflon tube. The wetting film, with the extracted analyte, was then eluted with 100 mul acetonitrile and the analyte determined spectrophotometrically at 546 nm. Important optimized parameters were the selection of wetting film and elution solvents, the flow rate, the length and diameter of the extraction coil and the conditions for the formation of the ion paired chelate. Cr(III) was previously oxidized to Cr(VI) and calculated as the difference between total Cr and Cr(VI). An enrichment factor of 25 and a detection limit of 2.0 mug l(-1) Cr(VI) were achieved with a sampling frequency of 17 h(-1). The calibration curve was linear up to 100 mug l(-1) Cr(VI) (r = 0.999). The relative standard deviations were 2.8 and 2.0% at the 25 and 100 mug l(-1) levels.

Titration without Mixing or Dilution: Sequential Injection of Chemical Sensing Membranes.

A novel, miniaturized titration was developed using beads 35 µm in diameter as semisolid aqueous titrant retained in a nonaqueous sample stream. Agarose beads with internally bound pH indicator served as a pH sensing membrane material swollen with aqueous NaOH titrant. The indicator monitored the remaining titrant within the agarose beads during perfusion with H(2)SO(4) in 1-butanol samples. Irreversible reaction of 2 mg bead layers was made possible by automated packing and disposal in a flow cell. This strategy substituted membrane advantages for the burdens of mixing and unnecessary dilution under laminar flow conditions. The agarose environment was conditioned with NaCl to tolerate dissolved salt in the sample. Transmittance measurements were made via fiber optics through FEP PTFE optical windows. A simple inverse relationship held between endpoint volume and acid concentration so that calibration curves were linear, R(2) = 0.9980.

Coaxial flow mixer for real-time monitoring of cellular responses in flow injection cytometry.

Improved time resolution of kinetic cellular events in flow cytometry is demonstrated by using a coaxial flow-mixing device integrated within a flow-injection (FI) system. The instrument is used in combination with a Becton Dickinson FACS Analyzer for on-line reagent addition, rapid sample mixing, and temperature control of cell suspensions. The coaxial flow device can instantaneously (< 60 ms) mix reagent and sample streams, allowing cytometric analysis of subsecond events to be performed. Kinetic measurements can be performed on the FACS analyzer in a variable time range of from 100 ms to 3 min. The system also allows the collection of unlimited cellular events at a specific incubation time point. Because the system operates continuously and no boost in core flow is required, disturbances of flow conditions are avoided. The capabilities of the flow injection cytometer have been demonstrated by the determination of internal [Ca2+]i mobilization in Jurkat T lymphocytes perfused internally with INDO-1 and stimulated by ionomycin.

Analytical strategies for the measurement of lithium in biological samples.

Therapeutic lithium levels in the treatment of manic depressive psychosis must be maintained in the range of 0.5-1.5 mM in the blood, which also contains 140 mM sodium. This paper reviews spectrophotometric, fluorometric, and ion-selective electrode (ISE) reagents and methods for achieving high lithium selectivity over sodium and their use in blood lithium measurement. These include aromatic organic reagents, crown ethers and amide ionophores. Crown ethers and cryptands provide the best lithium selectivity. A chromophoric small-cavity cryptand phenol exhibits greater than 4000:1 selectivity due to rigid configuration of a well preorganized binding site for lithium complexation. It is water soluble, making it easy to apply for blood analysis. Crown ethers with bulky groups inhibit formation of the 2:1 crown:sodium complex, while allowing formation of the 1:1 lithium complex. A PTM 14-crown-4 having a bulky pinane and subunits at the ethano bridge exhibits at least 10,000:1 selectivity for lithium in a flow-through optical sensor probe. Bulky crown ethers used in PVC membrane ion-selective electrodes exhibit lithium selectivities of 1-2000:1. Methods of evaluating selectivities are discussed, along with the correlation of solvent extraction of crown ether complexes and solvent membrane ISE selectivities.

Flow/sequential injection determination of gaseous ammonia with a glass diffusion denuder.

A new combination of a flow/sequential injection method for the analysis of ammonia has been developed. Gaseous ammonia is selectively absorbed in a phosphoric acid coated glass tube and determined with Berthelot reagents by flow injection. The combination of the gas diffusion denuder sampler with flow injection makes this method very sensitive and selective. The limit of detection of 0.15 mug NH(3).

A coaxial jet mixer for rapid kinetic analysis in flow injection and flow injection cytometry.

A simple coaxial jet mixer for rapid and efficient confluent mixing under laminar flow conditions (Re < 5) is described. This device demonstrates exceptional control of mixing between two laminar streams by creating shear forces due to variable flow velocities at the point of confluence. It is suitable for flow injection and cytometric analyses of rapid kinetic events which require contact mixing of two solutions and subsecond measurements of the evolving reaction. This apparatus was devised for flow injection cytometry as performed on a Becton Dickinson FACS Analyzer. Under normal cytometric conditions and at a sample introduction rate of 60 microL/min, the laminar jet mixer is capable of complete mixing of two solutions within 55 ms. Kinetic measurements can be performed on the FACS Analyzer in a variable time range of 100 ms to 3 min with 14-30 ms temporal resolution of the studied event. Since no boost in core flow is required, potential spectral distortions due to core flow variations are eliminated. This coaxial jet mixer can be easily constructed and employed on a variety of cytometers as well as conventional flow injection analysis systems, since it is an effective mixer under most flow conditions.

Detection of cocaine and its polar transformation products and metabolites in human urine.

This paper describes a method for the analysis of thermal degradation compounds generated from cocaine during the smoking process, together with chemical and enzymatic biotransformation products which, by virtue of their polarity, are not recovered by existing analytical procedures. The method employs cation exchange solid phase extraction coupled with gas chromatography/mass spectrometry. Compounds identified in urine from subjects of cocaine-related death included cocaine, benzoylecgonine, ecgonine methyl ester and ecgonine, which were measured quantitatively, and ecgonidine, ecgonidine methyl ester, ecgonine ethyl ester, ethyl benzoylecgonine, norcocaine, benzoylnorecgonine, cinnamoylcocaine, and cinnamoylecgonine. The concentrations of ecgonine (0-104 micrograms/ml) and ecgonine methyl ester (0-177 micrograms/ml) were substantial and averaged about one tenth the concentrations of benzoylecgonine (0-1074 micrograms/ml) and cocaine (0-1221 micrograms/ml). These and several of the other compounds identified will be valuable markers for cocaine use, in degraded samples and for indicating the route of administration.

Fast kinetic measurements and on-line dilution by flow injection cytometry.

An improved flow injection cytometry (FIC) system suitable for fast cellular kinetic measurements and on-line dilution is described. The instrument allows for measurements from 1.2 s (+/- 0.05 s) after the initiation of mixing, up to any time thereafter. Crucial factors in determining fast kinetic measurements, such as the displacement volume for sample introduction, rate of transport to cytometric interrogation point, and the mixing speed are evaluated and discussed. By varying the volume aspirated, this instrument can facilitate the dilution of cells and/or reagent over a range of one order of magnitude. The fast kinetic and on-line dilution capabilities were demonstrated by on-line staining of DNA in trout erythrocytes with 4',6-diamidino-2-phenylindole (DAPI). The utility of the instrument for measurement of enzyme kinetics was illustrated using human lymphocytes, measuring the glutathione S-transferase (GST) catalyzed reaction between monochlorobimane (MCB) and glutathione (GSH).

Flow injection titration-linear or logarithmic.

A simple and rapid Continuous Flow Injection Titration method utilizing a pretitration step was developed to automate the assay of high sulfuric acid concentrations over a narrow range in an oil refinery stream. A Coulometric Flow Injection Titration method was developed to perform Karl Fisher titration. Multiple functions of the mixing chamber in these FIA titrations such as for pretitration dilution, and reduction of viscosity are discussed. The cell also serves as the detector. Appropriate combining of these functions allows FIA titrations to become a powerful tool in automating routine assays.

An apparatus for the determination of volatile analytes by stopped-flow injection analysis using an integrated fiber optic detector.

A new method for the analysis of volatile analytes using a stopped-flow injection system originating from either a gas or liquid phase has been developed. It uses an integrated fiber optic detector which also serves as a reactor. This system combines the advantages of gas diffusion and stopped-flow, making the overall assay very sensitive. Both gas streams and aqueous solutions containing ammonia were analyzed. The limits of detection are 40 ppb for gas phase analysis and 1.0 ppm for aqueous phase analysis.