Analytical and Bioanalytical Chemistry (v.395, #2)

Explosives analysis by David S. Moore; John V. Goodpaster (245-246).
is presently a technical staff member in the Shock and Detonation Physics Group of Los Alamos National Laboratory. His fields of interest cover the application of optimal quantum control methods for condensed phase chemistry (explosive initiation) and dynamic trace detection. He is a Fellow of the American Physical Society (2004), an honor received for breakthroughs in the use of nonlinear optical and ultrafast spectroscopies to understand the behavior of molecules under shock compression. He was a Los Alamos National Laboratory Director Funded Postdoctoral Fellow (1980–1981) and an Alexander von Humboldt Fellow (Essen 1993–1994). is currently an assistant professor in the Forensic and Investigative Sciences (FIS) Program at Indiana University Purdue University Indianapolis (IUPUI). He teaches in the graduate program in the areas of alcohol and drug analysis as well as trace evidence. Ongoing research in his laboratory includes designing new techniques for explosives analysis as well as instrumental and statistical association of trace evidence. Current collaborations include projects with the Indiana State Police related to explosives, fire debris, and fiber evidence. Lastly, a federally funded project on the chemical compounds sensed by explosive-detecting canines is under way.

Terahertz spectroscopy techniques for explosives detection by Megan R. Leahy-Hoppa; Michael J. Fitch; Robert Osiander (247-257).
Spectroscopy in the terahertz frequency range has demonstrated unique identification of both pure and military-grade explosives. There is significant potential for wide applications of the technology for nondestructive and nonintrusive detection of explosives and related devices. Terahertz radiation can penetrate most dielectrics, such as clothing materials, plastics, and cardboard. This allows both screening of personnel and through-container screening. We review the capabilities of the technology to detect and identify explosives and highlight some of the critical works in this area.
Keywords: Terahertz; Far-infrared; Spectroscopy; Explosive

Laser-based standoff detection of explosives: a critical review by Sara Wallin; Anna Pettersson; Henric Östmark; Alison Hobro (259-274).
A review of standoff detection technologies for explosives has been made. The review is focused on trace detection methods (methods aiming to detect traces from handling explosives or the vapours surrounding an explosive charge due to the vapour pressure of the explosive) rather than bulk detection methods (methods aiming to detect the bulk explosive charge). The requirements for standoff detection technologies are discussed. The technologies discussed are mostly laser-based trace detection technologies, such as laser-induced-breakdown spectroscopy, Raman spectroscopy, laser-induced-fluorescence spectroscopy and IR spectroscopy but the bulk detection technologies millimetre wave imaging and terahertz spectroscopy are also discussed as a complement to the laser-based methods. The review includes novel techniques, not yet tested in realistic environments, more mature technologies which have been tested outdoors in realistic environments as well as the most mature millimetre wave imaging technique. Figure Standoff detection and identification is one of the most wanted capabilities
Keywords: Standoff detection; Optical sensors; Laser spectroscopy; Explosives detection; Review; Trace detection

Photonic sensor devices for explosive detection by Ulrike Willer; Wolfgang Schade (275-282).
For the sensitive online and in situ detection of gaseous species, optical methods are ideally suited. In contrast to chemical analysis, no sample preparation is necessary and therefore spectroscopic methods should be favorable both in respect of a fast signal recovery and economically because no disposal is needed. However, spectroscopic methods are currently not widely used for security applications. We review photonic sensor devices for the detection of explosives in the gas phase as well as the condensed phase and the underlying spectroscopic techniques with respect to their adaptability for security applications, where high sensitivity, high selectivity, and a low false-alarm rate are of importance. The measurements have to be performed under ambient conditions and often remote handling or even operation in standoff configuration is needed. For handheld and portable equipment, special attention is focused on the miniaturization and examples for already-available sensor devices are given.
Keywords: Photonic sensors; Explosives; Instrumentation; Security applications

Laser-induced breakdown spectroscopy for detection of explosives residues: a review of recent advances, challenges, and future prospects by Jennifer L. Gottfried; Frank C. De Lucia Jr; Chase A. Munson; Andrzej W. Miziolek (283-300).
In this review we discuss the application of laser-induced breakdown spectroscopy (LIBS) to the problem of detection of residues of explosives. Research in this area presented in open literature is reviewed. Both laboratory and field-tested standoff LIBS instruments have been used to detect explosive materials. Recent advances in instrumentation and data analysis techniques are discussed, including the use of double-pulse LIBS to reduce air entrainment in the analytical plasma and the application of advanced chemometric techniques such as partial least-squares discriminant analysis to discriminate between residues of explosives and non-explosives on various surfaces. A number of challenges associated with detection of explosives residues using LIBS have been identified, along with their possible solutions. Several groups have investigated methods for improving the sensitivity and selectivity of LIBS for detection of explosives, including the use of femtosecond-pulse lasers, supplemental enhancement of the laser-induced plasma emission, and complementary orthogonal techniques. Despite the associated challenges, researchers have demonstrated the tremendous potential of LIBS for real-time detection of explosives residues at standoff distances. Figure This review discusses the application of laser-induced breakdown spectroscopy (LIBS) to the problem of explosive residue detection. LIBS offers the capability for real-time, standoff detection of trace amounts of residue explosives on various surfaces
Keywords: Explosives detection; Laser-induced breakdown spectroscopy; Double-pulse LIBS; Chemometric analysis

Current trends in the detection of peroxide-based explosives by Raychelle M. Burks; David S. Hage (301-313).
The increased use of peroxide-based explosives (PBEs) in criminal and terrorist activity has created a demand for continued innovation in the detection of these agents. This review provides an update to a previous 2006 review on the detection of PBEs, with a focus in this report on luminescence and fluorescence methods, infrared and Raman spectroscopy, mass spectrometry, and electrochemical techniques. Newer developments in gas chromatography and high performance liquid chromatography methods are also discussed. One recent trend that is discussed is an emphasis on field measurements through the use of portable instruments or portable assay formats. An increase in the use of infrared spectroscopy and mass spectrometry for PBE analysis is also noted. The analysis of triacetone triperoxide has been the focus in the development of many of these methods, although hexamethylene triperoxide diamine has received increased attention in PBE detection during the last few years.
Keywords: Peroxide-based explosives; Triacetone triperoxide; Hexamethylene triperoxide diamine; Explosives analysis

Polarization orientation dependence of the far infrared spectra of oriented single crystals of 1,3,5-trinitro-S-triazine (RDX) using terahertz time–domain spectroscopy by V. H. Whitley; D. E. Hooks; K. J. Ramos; J. F. O’Hara; A. K. Azad; A. J. Taylor; J. Barber; R. D. Averitt (315-322).
The far infrared spectra of (100), (010), and (001)-oriented RDX single crystals were measured as the crystal was rotated about the axis perpendicular to the polarization plane of the incident radiation. Absorption measurements were taken at temperatures of both 20 K and 295 K for all rotations using terahertz time–domain spectroscopy. A number of discrete absorptions were found ranging from 10–100 cm−1 (0.3–3 THz). The absorptions are highly dependent on the orientation of the terahertz polarization with respect to crystallographic axes.
Keywords: IR spectroscopy/Raman spectroscopy; Organic compounds/trace organic compounds; Spectroscopy/instrumentation

Vibrational spectroscopy standoff detection of explosives by Leonardo C. Pacheco-Londoño; William Ortiz-Rivera; Oliva M. Primera-Pedrozo; Samuel P. Hernández-Rivera (323-335).
Standoff infrared and Raman spectroscopy (SIRS and SRS) detection systems were designed from commercial instrumentation and successfully tested in remote detection of high explosives (HE). The SIRS system was configured by coupling a Fourier-transform infrared interferometer to a gold mirror and detector. The SRS instrument was built by fiber coupling a spectrograph to a reflective telescope. HE samples were detected on stainless steel surfaces as thin films (2–30 μg/cm2) for SIRS experiments and as particles (3–85 mg) for SRS measurements. Nitroaromatic HEs: TNT, DNT, RDX, C4, and Semtex-H and TATP cyclic peroxide homemade explosive were used as targets. For the SIRS experiments, samples were placed at increasing distances and an infrared beam was reflected from the stainless steel surfaces coated with the target chemicals at an angle of ∼180° from surface normal. Stainless steel plates containing TNT and RDX were first characterized for coverage distribution and surface concentration by reflection–absorption infrared spectroscopy. Targets were then placed at the standoff distance and SIRS spectra were collected in active reflectance mode. Limits of detection (LOD) were determined for all distances measured for the target HE. LOD values of 18 and 20 μg/cm2 were obtained for TNT and RDX, respectively, for the SIR longest standoff distance measured. For SRS experiments, as low as 3 mg of TNT and RDX were detected at 7 m source–target distance employing 488 and 514.5 nm excitation wavelengths. The first detection and quantification study of the important formulation C4 is reported. Detection limits as function of laser powers and acquisition times and at a standoff distance of 7 m were obtained.
Keywords: Standoff infrared detection; Standoff Raman detection; High explosives; Explosive formulations

Passive standoff detection of RDX residues on metal surfaces via infrared hyperspectral imaging by Thomas A. Blake; James F. Kelly; Neal B. Gallagher; Paul L. Gassman; Timothy J. Johnson (337-348).
Hyperspectral images of galvanized steel plates, each containing a stain of cyclotrimethylenetrinitramine (RDX), were recorded using a commercial long-wave infrared imaging spectrometer. Demonstrations of passive RDX chemical detection at areal dosages between 16 and 90 µg/cm2 were carried out over practical standoff ranges between 14 and 50 m. Anomaly and target detection algorithms were applied to the images to determine the effect of areal dosage and sensing distance on detection performance for target RDX. The anomaly detection algorithms included principal component analysis, maximum autocorrelation factors, and principal autocorrelation factors. Maximum difference factors and principal difference factors are novel multivariate edge detection techniques that were examined for their utility in detection of the RDX stains in the images. A target detection algorithm based on generalized least squares was applied to the images, as well, to see if the algorithm can identify the compound in the stains on the plates using laboratory reflection spectra of RDX, cyclotetramethylenetetranitramine (HMX), and 2,4,6-trinitrotoluene (TNT) as the target spectra. The algorithm could easily distinguish between the nitroaromatic (TNT) compound and the nitramine (RDX, HMX) compounds, and, though the distinction between RDX and HMX was less clear, the mean weighted residuals identified RDX as the stain on the plate. Improvements that can be made in this detection technique are discussed in detail. As expected, it was found that detection was best for short distances and higher areal dosages. However, the target was easily detected at all distances and areal dosages used in this study.
Keywords: Standoff detection; Infrared hyperspectral imaging; RDX; HMX; TNT; Anomaly detection; Target detection; Principal component analysis; Maximum autocorrelation factors; Principal autocorrelation factors; Generalized least squares

Standoff detection of nitrotoluenes using 213-nm amplified spontaneous emission from nitric oxide by Bradley Arnold; Lisa Kelly; Jeffrey B. Oleske; Alexander Schill (349-355).
A method of standoff detection based on the observation of laser-induced fluorescence–amplified spontaneous emission (LIF-ASE) is described. LIF-ASE generates uniaxial intensity distributions of the observed fluorescence with the majority of intensity propagating along the excitation axis in both the forward and backward directions. The detection of bulk vapor at significant standoff distances is readily achieved. This method was used to detect NO directly and as a photoproduct after 213-nm excitation of 2-, 3-, and 4-nitrotoluene. The NO LIF-ASE spectra were studied as a function of buffer gas. These studies showed that the emission from different vibrational states was dependent upon the buffer gas used, suggesting that the populations of vibrational states were influenced by the environment. A similar sensitivity of the vibrational populations was observed when the different nitroaromatic precursors were used in nitrogen buffer gas. Such sensitivity to environmental influences can be used to distinguish among the different nitroaromatic precursors and facilitate the identification of the bulk vapor of these analytes.
Keywords: Amplified spontaneous emission; Nitrotoluenes; Standoff detection; Photofragmentation; Laser-induced fluorescence

Measurement of trace explosive residues in a surrogate operational environment: implications for tactical use of chemical sensing in C-IED operations by Roderick R. Kunz; Kerin Clow Gregory; Dennis Hardy; Jonathan Oyler; Stanley A. Ostazeski; Augustus Way Fountain III (357-369).
A campaign to measure the amount of trace explosive residues in an operational military environment was conducted on May 27–31, 2007, at the National Training Center at Fort Irwin, CA, USA. The objectives of this campaign were to develop the methods needed to collect and analyze samples from tactical military settings, to use the data obtained to determine what the trace explosive signatures suggest about the potential capabilities of chemical-based means to detect IEDs, and, finally, to present a framework whereby a sound understanding of the signature science can be used to guide development of new sensing technologies and sensor concepts of operation. Through our use of combined background and threat signature data, we have performed statistical analyses to estimate upper limits of notional sensor performance that is limited only by the spatial correlation of the signature chemicals to the threats of interest.
Keywords: Explosives detection; Background clutter; Trace residue; Improvised explosive device; TNT; RDX

Amperometric sensing of hydrogen peroxide vapor for security screening by John Benedet; Donglai Lu; Karel Cizek; Jeff La Belle; Joseph Wang (371-376).
Rapid detection of the hydrogen peroxide precursor of peroxide explosives is required in numerous security screening applications. We describe a highly sensitive and selective amperometric detection of hydrogen peroxide vapor at an agarose-coated Prussian-blue (PB) modified thick-film carbon transducer. The sensor responds rapidly and reversibly to dynamic changes in the level of the peroxide vapor, with no apparent carry over and with a detection limit of 6 ppbv. The remarkable selectivity of the PB-based screen-printed electrode towards hydrogen peroxide leads to effective discrimination against common beverage samples. For example, blind tests have demonstrated the ability to selectively and non-invasively identify concealed hydrogen peroxide in drinking cups and bottles. The attractive performance of the new microfabricated PB-based amperometric peroxide vapor sensor indicates great potential for addressing a wide range of security screening and surveillance applications. Figure Experimental setup (left) with three electrode electrochemical Hydrogen Peroxide sensor hanging above container of “unknown” liquid. Schematic (right) demonstrating fundamental principles of operation of the sensor.
Keywords: Hydrogen peroxide; Prussian-blue; Vapor sensor; Screen-printed electrodes; Explosives; Security screening

Absolute integrated intensities of vapor-phase hydrogen peroxide (H2O2) in the mid-infrared at atmospheric pressure by Timothy J. Johnson; Robert L. Sams; Sarah D. Burton; Thomas A. Blake (377-386).
We report quantitative infrared spectra of vapor-phase hydrogen peroxide (H2O2) with all spectra pressure-broadened to atmospheric pressure. The data were generated by injecting a concentrated solution (83%) of H2O2 into a gently heated disseminator and diluting it with pure N2 carrier gas. The water vapor lines were quantitatively subtracted from the resulting spectra to yield the spectrum of pure H2O2. The results for the ν6 band strength (including hot bands) compare favorably with the results of Klee et al. (J Mol. Spectrosc. 195:154, 1999) as well as with the HITRAN values. The present results are 433 and 467 cm-2 atm−1 (±8 and ±3% as measured at 298 and 323 K, respectively, and reduced to 296 K) for the band strength, matching well the value reported by Klee et al. (S = 467 cm−2 atm−1 at 296 K) for the integrated band. The ν1 + ν5 near-infrared band between 6,900 and 7,200 cm−1 has an integrated intensity S = 26.3 cm−2 atm−1, larger than previously reported values. Other infrared and near-infrared bands and their potential for atmospheric monitoring are discussed.
Keywords: Infrared; Fourier transform infrared; Quantitative; Band strengths; Hydrogen peroxide

Lewis acid–base interactions enhance explosives sensing in silacycle polymers by Jason C. Sanchez; Antonio G. DiPasquale; Anthony A. Mrse; William C. Trogler (387-392).
The high sensitivity of silole- and silafluorene-containing polymers for detecting organic nitro, nitrate, and nitramine explosives cannot be solely attributed to favorable analyte–polymer hydrophobic interactions and amplified fluorescence quenching due to delocalization along the polymer chain. The Lewis acidity of silicon in conjugated poly(silafluorene-vinylene)s is shown to be important. This was established by examining the 29Si NMR chemical shifts (Δ) for the model trimer fragment of the polymer CH3–silafluorene–(trans-C2H2)–silafluorene–(trans-C2H2)–silafluorene–CH3. The peripheral and central silicon resonances are up-field from a TMS reference at −9.50 and −18.9 ppm, respectively. Both resonances shift down-field in the presence of donor analytes and the observed shifts (0 to 1 ppm) correlate with the basicity of a variety of added Lewis bases, including TNT. The most basic analyte studied was acetonitrile and an association constant (K a) of 0.12 M−1 was calculated its binding to the peripheral silicon centers using the Scatchard method. Spin-lattice relaxation times (T 1) of 5.86(3) and 4.83(4) s were measured for the methyl protons of acetonitrile in benzene-d 6 at 20 °C in the absence and presence of the silafluorene trimer, respectively. The significant change in T 1 values further supports a binding event between acetonitrile and the silafluorene trimer. These studies as well as significant changes and shifts observed in the characteristic UV–Vis absorption of the silafluorene group support an important role for the Lewis acid character of Si in polymer sensors that incorporate strained silacycles. The nitro groups of high explosives may act as weak Lewis-base donors to silacycles. This provides a donor–acceptor interaction that may be crucial for orienting the explosive analyte in the polymer film to provide an efficient pathway for inner-sphere electron transfer during the electron-transfer quenching process. Figure
Keywords: Silafluorene; Explosives; Sensor; Luminescent; Polymers

A chloride-anion insensitive colorimetric chemosensor for trinitrobenzene and picric acid by Dae-Sik Kim; Vincent M. Lynch; Kent A. Nielsen; Carsten Johnsen; Jan O. Jeppesen; Jonathan L. Sessler (393-400).
A new receptor, the bisTTF-calix[2]thiophene[2]pyrrole derivative 3, has been prepared from the Lewis acid-catalyzed condensation of 2,5-bis(1-hydroxymethylethyl)thiopheno-TTF and pyrrole. This new system is found to form complexes with the electron-deficient guests, trinitrobenzene (TNB) and picric acid (PA), which serve as models for nitroaromatic explosives. The binding phenomenon, which has been studied in organic solution using proton nuclear magnetic resonance and absorption spectroscopies, results in an easy-to-visualize color change in chloroform that is independent of the presence of chloride anion, a known interferant for an earlier tetrakisTTF-calix[4]pyrrole TNB chemosensor. Support for the proposed binding mode comes from a preliminary solid state structure of the complex formed from TNB, namely TNB⊂3. A color change is also observed when dichloromethane solutions of chemosensor 3 are added to solvent-free samples of TNB, PA, and 2,4,6-trinitrotoluene supported on silica gel. Figure A new bis-tetrathiafulvalene calix[2]thiophene[2]pyrrole derivative has been prepared that gives rise to an easy-to-visualize color change in the presence of the model nitroaromatic explosives trinitrobenzene and picric acid.
Keywords: Calix[4]pyrroles; Charge transfer (CT); Colorimetric chemosensors; Explosive detection; Supramolecular chemistry; Tetrathiafulvalenes

Bioassays for bomb-makers: proof of concept by Suzanne C. Bell; Melissa Gayton-Ely; Corey M. Nida (401-409).
Clandestine bomb-makers are exposed to significant amounts of explosives and allied materials. As with any ingested xenobiotic substance, these compounds are subject to biotransformation. As such, the potential exists that characteristic suites of biomarkers may be produced and deposited in matrices that can be exploited for forensic and investigative purposes. However, before such assays can be developed, foundational data must be gathered regarding the toxicokinetics, fate, and transport of the resulting biomarkers within the body and in matrices such as urine, hair, nails, sweat, feces, and saliva. This report presents an in vitro method for simulation of human metabolic transformations using human liver microsomes and an assay applicable to representative nitro-explosives. Control and metabolized samples of TNT, RDX, HMX, and tetryl were analyzed using high-performance liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) and biomarkers identified for each. The challenges associated with this method arise from solubility issues and limitations imposed by instrumentation, specifically, modes of ionization.
Keywords: Explosives; Biomarkers; Metabolism; LC/MS/MS

The implementation of hand-held ion mobility spectrometers (IMS) requires the development and evaluation of miniature drift cells providing high sensitivity while maintaining reasonable resolution. This manuscript describes the construction of a miniature IMS designed for such an application and its characterization by evaluation of the detection limits and resolution of the system with seven explosive compounds including trinitrotoluene (TNT), cyclotrimethylenetrinitramine (RDX), pentaerythritol tetranitrate (PETN), 2,4,6-trinitrophenyl-N-methylnitramine (Tetryl), nitroglycerin (NG), 2,4-dinitrotoluene (2,4 DNT), and 2,6-dinitrotoluene (2,6 DNT). Miniature IMS Assembly
Keywords: Miniature IMS; Explosives; Capacitive-transimpedance amplifier; CTIA

Urea nitrate, an exceptionally easy-to-make improvised explosive: studies towards trace characterization by Tsippy Tamiri; Rinat Rozin; Nitay Lemberger; Joseph Almog (421-428).
Urea nitrate is a powerful improvised explosive, frequently used by terrorists in the Israeli arena. It was also used in the first World Trade Center bombing in New York in February 1993. It is difficult to identify urea nitrate in post-explosion debris, since only a very small fraction survives the blast. Also, in the presence of water, it readily decomposes to its original components, urea and nitric acid. It is suspected that post-blast debris of urea nitrate can be confused with ammonium nitrate, the main solid product of urea nitrate thermal decomposition. In a comprehensive study towards identification of urea nitrate in post-blast traces, a spectrophotometric technique for quantitative determination of urea nitrate was developed, and conditions were found for extraction and separation of un-exploded traces of urea nitrate with minimal decomposition. Nevertheless, out of 28 samples collected from a series of three controlled firings of urea nitrate charges, only one gave the typical adduct ion by liquid chromatography/mass spectrometry analysis. We found that urea nitrate can be extracted from solid mixtures to organic solvents by using Crown ethers as “host compounds.” The adducts thus formed are solid, crystalline compounds that can be characterized by microanalysis and spectroscopic techniques. Figure Adduct formation between urea nitrate and 18-crown-6
Keywords: Forensics; Improvised explosives; Post-blast; Urea nitrate; LC/MS; Crown ethers

The properties of native and oxidised graphene layered carbon nanofibres are compared, and their utilisation in enzyme biosensor systems using different immobilisation methods are evaluated. The efficient oxidation of carbon nanofibres with concentrated H2SO4/HNO3 is confirmed by Raman spectroscopy while the introduction of carboxylic acid groups on the surface of the fibres by titration studies. The oxidised fibres show enhanced oxidation efficiency to hydrogen peroxide, while at the same time they exhibit a more efficient and selective interaction with enzymes. The analytical characteristics of biosensor systems based on the adsorption or covalent immobilisation of the enzyme glucose oxidase on carbon nanofibres are compared. The study reveals that carbon nanofibres are excellent substrates for enzyme immobilisation allowing the development of highly stable biosensor systems. Figure Immobilization of proteins on carbon nanofibres
Keywords: Graphene layered carbon nanofibres; Oxidation; Covalent immobilisation; Adsorption; Biosensor

One of the main problems of anti-cancer therapy is an insufficient differentiation between normal and malignant cells by the known anti-proliferant agents. The antibody-directed enzyme prodrug therapy is a promising approach for a selective treatment of cancer, in which a non-toxic prodrug is enzymatically converted into a highly cytotoxic drug at the surface of malignant cells by a targeted antibody–enzyme conjugate. The transformations and the stability of a very promising novel prodrug and its corresponding cytotoxic derivative were now investigated in detail by high-performance liquid chromatography (HPLC)–mass spectrometry (MS). In order to determine the time-dependent DNA alkylation efficiency and the sequence selectivity of the novel compounds, DNA binding studies using direct electrospray–Fourier transform ion cyclotron resonance–MS (ESI–FTICR–MS) have been performed. These measurements were accompanied by HPLC analyses followed by MS of the separated species to confirm the results of the direct ESI–FTICR–MS measurements. The sites of DNA alkylation could be identified unambiguously by the mass spectrometric fragmentation pattern of the alkylated oligodeoxynucleotides as well as by the results of HPLC followed by MS. A combination of all techniques applied led to a better understanding of the mode of action of the new therapeutics and might be used for an estimation of the cytotoxicity of different prodrugs and drugs since the alkylation efficiency correlates with the bioactivity of the compounds in cell culture investigations. After enzymatic cleavage of the sugar moiety, the untoxic prodrug is converted rapidly into the corresponding highly cytotoxic drug that alkylates DNA with high efficiency
Keywords: DNA alkylation; Anti-cancer drug; Duocarmycins; HPLC–MS; Mass spectrometry; Prodrug

The introduction of specific molecules into live cells is a widely used approach to probe cellular mechanisms. Recently, we have reported on the sustained dosing of molecules into single cells via a microscopic diffusion port. Here we describe temporal ratiometry, a method to reconstruct intracellular concentration distribution of the delivered molecules as it varies in time during dosing. To characterize this method, we analyzed fluorescence intensity maps obtained during delivery of Lucifer Yellow CH, LY, a polar fluorophore into A7r5 vascular smooth muscle cells, normal rat kidney epithelial cells (NRKE), and MCF-7 human breast cancer cells. Temporal ratiometry indicates a linear increase in concentration of LY in these cells with a nearly uniform distribution during 20 min of delivery. The method cancels the effects of varying cell height and variable accessible volume on the measured intensities at different locations within the cell. Temporal ratiometry will be useful to estimate dynamic changes in intracellular concentration distributions and thus, facilitate the understanding of transport, binding, sequestration, and efflux of molecules introduced into cells.
Keywords: Temporal ratiometry; Dynamic concentration distribution; Diffusional microburet; Continuous intracellular dosing

Toxaphene analysis in Great Lakes fish: a comparison of GC-EI/MS/MS and GC-ECNI-MS, individual congener standard and technical mixture for quantification of toxaphene by Xiaoyan Xia; Bernard S. Crimmins; Philip K. Hopke; James J. Pagano; Michael S. Milligan; Thomas M. Holsen (457-463).
Toxaphene is considered to be a problematic organochlorine pollutant because of its bioaccumulation potential and persistence in aquatic environments. In this study, whole lake trout and walleye composites were used to evaluate two analytical techniques for total toxaphene and selected congener analysis. The efficacy of using gas chromatography electron ionization tandem mass spectrometry (GC-EI/MS/MS) and electron capture negative ionization mass spectrometry (GC-ECNI-MS) were compared. Although the sensitivity using GC-ECNI-MS was approximately five times greater than GC-EI/MS/MS, the latter provided more consistent inter-Parlar relative response factors (RRF). When using technical calibration mixtures, these results suggest a more accurate total toxaphene measurement was obtained using the GC-EI/MS/MS method. Total toxaphene concentrations in lake trout composites from both methods were highly correlated (R 2 = 0.985) with the MS/MS concentrations approximately half of those determined by ECNI, suggesting systematic high bias in toxaphene concentrations when measured using GC-ECNI.
Keywords: Toxaphene; Great Lakes; Gas chromatography coupled with electron ionization tandem mass spectrometry (GC-EI/MS/MS); Gas chromatography/electron capture negative ion mass spectrometry (GC-ECNI-MS); Fish tissue

A novel method based on solid-phase extraction was studied for the extraction of amitrole (3-amino-1,2,4-triazole), and its residue determination in apples has been developed. The samples were derivatized with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF). The derivatization conditions and the influence of elution composition on the separation were investigated. In pH 9.5 H3BO3–Na2B4O7 media, the reaction of amitrole with CNBF was complete at 60°C after 30 min. The separation of derivatized amitrole was achieved at room temperature within 13 min by gradient elution mode with cetyltrimethylammonium bromide in mobile phase as ion-pair reagent. The method correlation coefficient was 0.9996, in concentrations ranging from 1.66 to 415 mg L−1. The calculated recoveries of the proposed method were from 94.17% to 105.67%, and relative standard deviations were 1.57% to 6.44% in the application to the quantitative determination of amitrole in apples. The detection limit of amitrole was 0.10 mg L−1 with a signal-to-noise ratio of 3. Figure Residue determination of amitrole in apple by ion-pairing high-performance liquid chromatography
Keywords: Amitrole; Precolumn derivatization; 4-Chloro-3,5-dinitrobenzotrifluoride (CNBF); High-performance liquid chromatography; Apple

Application of external micro-spectrophotometric detection to improve sensitivity on microchips by Attila Gáspár; István Bácsi; Erika F. Garcia; Mihály Braun; Frank A. Gomez (473-478).
The goal of this work was to increase the sensitivity of a UV–Vis spectrophotometer by decreasing the background noise and lengthening the optical path. A microphotometer has been modified to precisely select very small parts of a microfluidic channel pattern of a chip and to measure light absorbance on a magnified area of the selected part of the channel. The viability of combining a projection microscope and a spectrophotometer for external absorbance measurements on disposable PDMS chips was studied. Besides the external direct detection above a microfluidic channel, the optical pathlength was lengthened by detecting in the region of the perpendicular exit port. Increasing the cross-sectional area of the zone of irradiation improved the signal-to-noise ratio and the limits of detection (LOD).
Keywords: Chip; Poly(dimethylsiloxane); External UV detection; Micro-spectrophotometer; Flow injection; Microfluidics/microfabrication; UV/Vis

Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) has been widely used as a biomarker of oxidative DNA damage. Measurements of 8-OHdG in urinary samples are challenging owing to the low level of 8-OHdG and the complex matrix. In this study, a novel molecularly imprinted polymer (MIP) monolithic column was synthesized with guanosine as a dummy template which was used as the medium for in-tube solid-phase microextraction (SPME). In-tube SPME coupled with HPLC/UV detection for extraction and determination of urinary 8-OHdG was developed. The synthesized MIP monolithic column exhibited high extraction efficiency owing to its greater phase ratio with convective mass transfer and inherent selectivity. The enrichment factor for 8-OHdG was found to be 76 and the limits of detection and quantification of the method for urinary samples were 3.2 nmol/L (signal-to-noise ratio 3) and 11 nmol/L (signal-to-noise ratio 10), respectively. The MIPs selectivity also made the sample preparation procedure and chromatographic separation much easier. The linear range of the proposed method was from 0.010 to 5.30 μmol/L (r = 0.9997), with a relative standard deviation of 1.1–6.8%, and the recovery for spiked urine samples was 84 ± 3%. The newly developed method was successfully applied to determine urinary samples of healthy volunteers, coking plant workers, and cancer patients. The 8-OHdG level in cancer patients was significantly higher than that in healthy people.
Keywords: Molecularly imprinted monolith; In-tube solid-phase microextraction; 8-Hydroxy-2′-deoxyguanosine; Urine

Simple and sensitive colorimetric detection of cysteine based on ssDNA-stabilized gold nanoparticles by Zhang Chen; Shenglian Luo; Chengbin Liu; Qingyun Cai (489-494).
In this paper, we demonstrate a simple and sensitive colorimetric detection of cysteine based on the cysteine-mediated color change of ssDNA-stabilized gold nanoparticles (AuNPs). Cysteine is capable of absorbing onto AuNPs surfaces via the strong interaction between its thiol group and gold. ssDNA molecules which stabilize AuNPs against salt-induced aggregation are removed away by cysteine encapsulation on the AuNPs surfaces, resulting in a characteristic color change of AuNPs from red to blue as soon as salt is added. The ratio of absorptions at 640 to 525 nm (A 640/A 525) is linear dependent on the cysteine concentration in the range from 0.1 to 5 μM. Furthermore, amino acids other than cysteine cannot mediate the color change under the identical conditions due to the absence of thiol groups, suggesting the selectivity of the proposed method toward cysteine. The employment of complicated protocols and sophisticated processes such as the preparation of modified AuNPs are successfully avoided in design to realize the simple and low-cost cysteine detection; and the high sensitivity and low cost of the method is favorable for practical applications. Figure In the presence of cysteine, cysteine binds to the AuNPs surface via Au-S bond, spontaneously driving ssDNA molecules away from the nanoparticles, which leads to the AuNPs aggregation under the condition of NaCl introduction, and the corresponding color change from red to blue. However, the presence of other amino acids results in no color change due to the absence of thiol groups.
Keywords: Gold nanoparticles; ssDNA; Cysteine; Colorimetric detection

Characterization of steroidal saponins in saponin extract from Paris polyphylla by liquid chromatography tandem multi-stage mass spectrometry by Shuli Man; Wenyuan Gao; Yanjun Zhang; Xinghua Jin; Chaoyi Ma; Xianxiao Huang; Qingyun Li (495-505).
Rhizoma Paridis saponins are bioactive steroidal saponins derived from Paris polyphylla. Optimization of the ionization process was performed with electrospray ionization tandem mass spectrometry in both positive and negative-ion modes. Negative-ion ESI was adopted for generation of the precursor deprotonated molecules to achieve the best ionization sensitivity for the analytes. Positive ionization was used to choose the most abundant fragment ion. Furthermore, according to the characteristic fragmentation behavior of known steroidal saponins isolated from this plant (polyphyllin D, formosanin C, gracillin, Paris H, Paris VII, and dioscin), 23 constituents were structurally characterized on the basis of their retention time and ESI analyses, including four pairs of naturally occurring isomers. Five of these 23 constituents were new compounds. The analytical method of LC–MS n in positive and negative-ion modes has been developed for the direct structural elucidation of steroid saponins of this kind in plant extracts.
Keywords: Electrospray ionization tandem mass spectrometry; Fragmentation pathways; HPLC-MS; Paris polyphylla ; Steroidal saponin

In order to study the effect of the nature and the length of the spacer, three mixed 10-undecenoate/phenylcarbamate derivatives of β-cyclodextrin have been prepared and linked to allylsilica gel by means of a radical reaction. The chiral recognition ability of the resulting materials, when used as liquid chromatography chiral stationary phases (CSPs), was evaluated using heptane and either 2-propanol or chloroform as organic mobile-phase modifiers. A large variety of racemic compounds have been separated successfully on these CSPs (mainly pharmaceuticals and herbicides). Optimization of these separations was discussed in terms of mobile-phase composition and structural patterns of the injected analytes. The efficiencies of the three prepared materials were compared to those of previously described perphenylated-β-cyclodextrin column and to analogous cellulose derivative-based CSPs. Schematic illustration of the b-cyclodextrin/mandelic acid inclusion complex
Keywords: Chiral stationary phases; Cyclodextrin; 10-Undecenoate/phenylcarbamate-β-CD; HPLC; Enantiomeric resolutions; Pharmaceuticals; Herbicides

Depolymerization study of sodium hyaluronate by flow field-flow fractionation/multiangle light scattering by Ji Hye Kwon; Euijin Hwang; Il-Hwan Cho; Myeong Hee Moon (519-525).
Thermal depolymerization of ultrahigh-molecular-weight (UHMW) sodium hyaluronate (NaHA) was studied systematically by using frit-inlet asymmetrical flow field-flow fractionation/multiangle light scattering/differential refractive index (FI-AFlFFF/MALS/DRI). FI-AFlFFF was utilized for the size separation of NaHA samples which had been thermally degraded for varied treatment times, followed by light-scattering detection to determine MW and structural information of degraded NaHA products. Analysis of NaHA products showed time-dependent depolymerization of raw molecules into smaller-MW components, as well as unfolding of compact structures of UHMW NaHA. To determine whether the observed decrease in MW of sodium hyaluronate originated from the chain degradation of UHMW molecules or from dissociation of entangled complex particles that may have been formed by intermolecular association, narrow size fractions (1 × 107–6 × 107 and >6 × 107 MW) of NaHA molecules were collected during FlFFF separation and followed by thermal treatment. Subsequent FI-AFlFFF/MALS analysis of collected fractions after thermal treatment suggested that the ultrahigh-MW region (>107 Da) of NaHA is likely to result from supermolecular structures formed by aggregation of large molecules.
Keywords: Flow field-flow fractionation; Multiangle light scattering; Sodium hyaluronate; Depolymerization; Biopolymers; Separations/instrumentation

A high-performance liquid chromatographic (HPLC) method with fluorescence detection for the quantification of vancomycin in human plasma was developed and validated. The method includes an extraction of vancomycin by deproteinization with acetonitrile. The analyses were carried out at 258 nm as the emission wavelength while exciting at 225 nm on a reversed-phase column (30 cm × 4 mm i.d. × 10 µm Waters Associates μBondapak C18) using a mobile phase composed of methanol and phosphate buffer at pH 6.3. Vancomycin was quantitatively recovered from human plasma samples (>96%) with high values of precision. The separation was completed within 27 min. The calibration curve was linear over the range from 5 to 1,000 ng/mL with the detection and quantification limits of 2 ng/mL and 5 ng/mL, respectively. This method is suitable for the routine assay of plasma samples. Figure The effect of the deproteinization solvent on the signal of the interference peak at retention time of 15.0 min. The peak which interferes with the peaks of Erythromycin and Vancomycin has been disappeared by using 2 mL acetonitrile as the deproteinization solvent.
Keywords: Vancomycin; Erythromycin; Internal standard; Validation; Fluorescence; Deproteinization

An LC method for monitoring medium-chain fatty acid permeation through CaCo-2 cell monolayers by Kazuhiro Yamamoto; Hideki Hakamata; Akira Yamaguchi; Fumiyo Kusu (533-538).
A simple method was developed for monitoring the permeation of medium-chain fatty acids of C8 (octanoic acid) and C10 (decanoic acid) through CaCo-2 cell monolayers by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The detection was made based on the electrochemical reduction prepeak of quinone caused by acids, requiring the fabrication of a two-channel HPLC-ECD system. In one channel, acetonitrile–water (7:3, v/v) was used as a mobile phase to separate acids by a C30 column. In the other channel, acetonitrile–water (7:3, v/v) containing 6 mmol/L 3,5-di-t-butyl-1,2-benzoquinone and 20 mmol/L LiClO4 was used as a quinone solution to detect acids by an electrochemical cell with a glassy carbon working electrode. In this HPLC-ECD system, eluted acids were mixed with the quinone solution in a post column fashion to obtain current signals caused by acids. The peak area was found to be linearly related to the acid amount ranging from 25 to 1,000 pmol (r > 0.992). The detection limits of octanoic acid and decanoic acid were 7.5 and 8.8 pmol, respectively. Octanoic acid and decanoic acid spiked into cell culture media samples were extracted with acetonitrile and their recoveries were more than 89.5% with an RSD of less than 8.2%. This method was applied to the permeation experiment of octanoic acid and decanoic acid with CaCo-2 cell monolayers formed on the Transwell® system.
Keywords: Octanoic acid; Decanoic acid; Medium-chain fatty acid; HPLC-ECD; CaCo-2