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Synopses of significant analytical articles from other publications
Determining GABA in spinal fluid Changes in the concentration of γ-aminobutyric acid (GABA) in cerebro spinal fluid have been reported in several neuropsychiatrie disorders. GABA is a major inhibitory neurotransmitter in the mammalian cen tral nervous system. Carolyn S. Shuck and colleagues at the Indiana University School of Medicine and the Veterans Ad ministration Medical Center have devel oped a radioligand competitive binding procedure that uses a mutant strain of Pseudomonasfluorescensto detect GABA in human cerebrospinal fluid. The method is based on competition between labeled and unlabeled GABA and involves an energy-dependent reaction that results in an irreversible fixing of ra dioactivity by the cells. In a double-blind study, GABA concentrations were mea sured by the Pseudomonas method, by a standard radioligand competitive binding assay using rat brain membrane, and by HPLC. The results obtained with the bac terial method correlated well with those obtained using the standard methods. The researchers also found that the bacte rial method is highly reproducible and is not affected by other naturally occurring compounds that are found in higher con centrations than GABA in cerebrospinal fluid or that were suspected to interfere with the neurotransmitter in a binding as say. A detection limit of 5 nM GABA was achieved. (Anal. Biochem. 1 9 9 5 , 225, 283-85 and 286-90)
Characterizing ibuprofen binding Protein binding is significant in the trans port and release of many drugs in blood because these interactions can influence the biological distribution, as well as ex cretion, therapeutic activity, and toxic ity, of the drug. Ibuprofen, a common non steroidal anti-inflammatory agent, exists in two enantiomeric forms, and there is ev idence that the two forms may bind differ ently to human serum albumin (HSA). David S. Hage and colleagues at the Uni-
versity of Nebraska and McGill University (Canada) have used zonal elution and high-performance affinity chromatogra phy to study the binding characteristics of R- and S- ibuprofen with HSA They injected small amounts of each enantiomer onto an HSA column while a known concentration otR- or S-ibuprofen was applied to the column through the mo bile phase. By examining how the mobilephase concentration of ibuprofen affected the retention of the injected solute, the researchers gained information on the type of competition between the two spe cies. They then used these data to deter mine the number of binding regions for each enantiomer and the corresponding association constants. The results indicate that R- and S-ibuprofen have one com mon binding site on the immobilized HSA column and that S-ibuprofen has at least one other major binding region. (J. Chromatogr. A 1995, 693, 23-32)
Glutathione's effect on serotonin oxidation Researchers have discovered that oxidized forms of 5-hydroxytryptamine (5-HT or serotonin) and 5-hydroxytryptophan are present in the cerebrospinal fluid of Alzhe imer's patients but not in that of agematched controls. This evidence, together with biochemical and histopathological abnormalities observed in the Alzheimer's brain, suggest that abnormal oxidative transformations of 5-HT and other endoge nous central indoles might result in the formation of toxic metabolites that contrib ute to the system degeneration that oc curs in Alzheimer's disease. To clarify how these reactions might occur in serotoner gic nerve terminals in the presence of glu tathione (GSH), Glenn Dryhurst and co workers at the University of Oklahoma studied the electrochemically driven and tyrosinase + 02-mediated oxidation of 5-HT using LC and cyclic voltammetry. They found that in the absence of GSH, 5-HT is oxidized to a C4 carbocation intermediate that reacts with 5-HT to give 5-5'-dihydroxy-4,4-bitryptamine and a number of other C-C and ether-linked dimers and oligomers, or with water to form tryptamine-4,5-dione. When GSH is present, the normal oxidation reaction is
partially diverted and results in formation of 4-S-glutathionyl-5-hydroxytryptamine. The researchers suggest that this product may be a useful analytical marker for un usual oxidation reactions of 5-HT in the Alzheimer's brain. (J. Electroanal. Chem. 1995, 382, 41-51)
Studying noncovalent interactions with CE/MS and CE/MS/MS The noncovalent complex of the immunophilin FKBP with the immunosuppres sive drug FK506 disrupts calcium-depen dent binding to the protein phosphatase calcineurin, whereas the FKBP complex and a related ligand, rapamycin (RM), block a family of calcium-independent sig naling pathways triggered byribosomalki nases. Jack D. Henion and colleagues at Cornell University investigated the possibil ity of using CE coupled with MS, a combi nation that provides high separation effi ciency with an orthogonal separation mechanism, to expand earlier studies on the analysis of immunophilin complexes. Samples were prepared using aqueous pH-neutral conditions and were analyzed by on-line CE/MS under selected ion mon itoring conditions and by CE/MS/MS under selected reaction monitoring condi tions. Target complexes were separated by CE with MS detection of the individual complexes between FKBP and FK506 [hFKBP+FK506+7H]7* as well as FKBP and RM [hFKBP+RM+7H]7+. The re searchers also analyzed a mixture of FK506 and RM in the presence of FKBP and observed a 9:1 ratio of ion current abundances between the RM and FK506 complexes, confirming results reported in other studies. They suggest that these results indicate the usefulness of CE/MS and CE/MS/MS for studying noncovalent interactions of biologically important macromolecules under physiological con ditions. (J. Am. Soc. Mass Spectrom. 1995, 6,85-90)
LC of immunosuppressants Cyclosporin A is used during and after transplant surgery to suppress the pa tient's immune reaction to and rejection of a donated organ. Therapeutic drug moni toring is an important part of the treat-
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ment protocol, but immunoassay methods in common use for monitoring cyclosporin A are not always accurate and LC with spectrophotometric detection lacks sensitivity. Masatoki Katayama and coworkers at the Meiji College of Pharmacy, Tachikawa Kyousai Hospital, Ichikawa General Hospital, and Keio University (japan) have developed a chemiluminescence detection method for LC determination of cyclosporin A. The chemiluminescence reaction is based on aryl oxalate reaction with the amide bond of the drugs. Cyclosporin A was separated on a C8 column using a methanol-water mobile phase containing 0.1 mol/L hydrogen peroxide. The eluent was mixed with an aryl oxalate and sulforhodamine 101 dye. The researchers were able to determine cyclosporin A from the low-nmol/L range to 0.02 mol/L with an RSD of 5.1% for micromolar concentrations; recovery from spiked serum samples was 89.1%. For both serum and wholeblood samples, the results obtained using the LC chemiluminescence method correlated well with those obtained using a fluorescence polarization immunoassay and an LC method with UV detection. (Anal. Chim. Acta 1995, 303, 333-40)
Detecting single molecules with SECM In recent years researchers have used several techniques to detect single molecules or ions in different environments. These are largely based on the observation of fluorescence from the molecule of interest and provide information about the molecule's environment and energy levels. Taking a different approach, Allen J. Bard and Fu-Ren F. Fan at the University of Texas, Austin, used a scanning electrochemical microscope to achieve single-molecule detection of an electroactive molecule in solution as it repeatedly underwent electron-transfer reactions at an electrode held a small distance from a counter electrode. Initially they trapped a small ( ~ 10-18 cm3) volume of a dilute solution of [(trimethylammonio) methyl] ferrocene (Cp2FeTMA+) between an ultramicroelectrode tip with a diameter of ~ 15 nm and a conductive substrate. The solution concentration was chosen so that, on average, only a single molecule resided in the trapped volume. The tip-substrate dis290 A
tance was adjusted to ~ 10 nm with the scanning electrochemical microscope and the oxidation of Cp2FeTMA+ to Cp2FeTMA2+ was carried out. The response was stochastic and anodic current peaks were observed as the molecule moved in and out of the tip-substrate gap. The researchers performed similar experiments with a solution that contained two redox species, ferrocene carboxylate (Cp2FeCOCr) and Os(bpy)f+. (Science 1995,267,871-74)
Microelectrode for dissolved redox species Membrane microelectrodes have become an important tool for scientists interested in elucidating natural microbial and biogeochemical processes. However, because the signal for amperometric electrodes is measured as a current at fixed potential, the electrodes are typically limited to a single analyte each. Scanning voltage while measuring current in a normal voltammetric experiment has not been done. George W. Luther, III, and Paul J. Brendel of the University of Delaware reasoned that a device capable of determining the concentration changes of several redox species at a time in suboxic sedimentary environments would be of great use to researchers interested in studying natural biogeochemical processes. The developed a solid-state voltammetric gold amalgam microelectrode to measure the dissolved redox species, 0 2 , S(II), Fe, and Mn, in the porewaters of marine and freshwater sediments. The use of fastscan voltammetric methods allowed simultaneous measurement of all redox species during one potential scan. Between scans, the electrode was electrochemically conditioned while it was deployed in water and sediments, allowing repeated use. The researchers obtained depth profiles at millimeter resolution for the redox species in a Delaware salt marsh and obtained results that are consistent with the known biogeochemical cycling of the target redox species. (Environ. Sci. Technol. 1995, 29, 751-61)
A new molecular ion source Primary ions are often used to transfer large organic molecules from the solid into the gaseous phase for mass spectrometric analysis. Because of the relatively small pri-
Analytical Chemistry, Vol. 67, No. 9, May 1, 1995
mary ion rate, a time-of-flight mass spectrometer must be used for analysis. Helmut Voit and colleagues at the Physikalisches Institut der Universifât Erlangen-Niirnberg (Germany) have investigated whether a simple and inexpensive primary ion source based on spontaneous desorption (the release of atomic and molecular ions from a sample without bombarding it with an external beam of particles or photons) could be used for the keV ion-induced mass spectrometric analysis of organic molecules. The researchers developed a molecular-ion source that consists of a grid in front of the plane sample and a conversion target for molecular ions. The source is particularly well suited for analyses of organic molecules with masses up to 1000 u because the average mass of ions delivered to the source can be chosen to be relatively large provided that a suitable conversion target (such as Csl) is used. However, only negative-ion spectra can be obtained from a sample because electrons are needed as trigger particles for the TOF measurement. (Rapid Commun. Mass Spectrom. 1995, 9, 209-12)
Capillary source for electrohydrodynamic MS Electrohydrodynamic MS (EHMS), like FDMS and ESIMS, is a soft ionization method. Unlike the other two methods, it is not widely used because signal stability is poor and because the release of ions under vacuum conditions demands a solvent with low vapor pressure, usually glycerol, limiting applications to compounds that are soluble in glycerol. Building on the early finding that ion source performance for EHMS improves when the sample liquid flow to the capillary emitter is discontinuous, Th. Diilcks and F. W. Rôllgen of the University of Bonn (Germany) designed a new ion source that operates without an external sample supply system. At a sample consumption rate of a few microliters per hour, the metal capillary of a conventional EHMS ion source holds enough sample to maintain ion emission for more than 1 h. The researchers eliminated the syringe from their design and used the metal capillary as both the field anode or cathode and as the sample reservoir, mounting it on a standard FD emitter carrier for compatibility with FD systems. The ion source prevented the emission of large droplets and allowed the acquisition of EH mass spectra for compounds with glycerol solubility greater than 0.5 mol/L, including sugars, amino acids, peptides, and
