Analytica Chimica Acta (v.661, #1)

Ionic liquids in analytical chemistry by Ping Sun; Daniel W. Armstrong (1-16).
Ionic liquids (ILs) are composed entirely of ions and they possess fascinating properties, including low volatility, tunable viscosity and miscibility, and electrolytic conductivity, which make ILs unique and useful for many applications in chemical analysis. The dramatic increase in the number of publications on ILs is indicative of the tremendous interest in this field from analytical chemists. This review summarizes recent efforts in the major subdisciplines of analytical chemistry, including extractions, gas chromatography, liquid chromatography, capillary electrophoresis, mass spectrometry, electrochemistry, sensors, and spectroscopy.
Keywords: Ionic liquids; Chemical analysis; Chromatography; Spectroscopy; Electrochemistry;

A dry ashing procedure is developed for the determination of As in organic rich matrices such as wheat flour, lichen and tobacco leaves. The volatility of As during dry ashing is avoided by the addition of palladium nitrate [Pd(NO3)2]. The recovery of both As(III) and As(V) is found to be near quantitative. The residue after dry ashing is dissolved in nitric acid (HNO3) and analysed by inductively coupled plasma-mass spectrometry (ICP-MS). The process blank and limit of detection (LOD) are 11 and 6.6 ng g−1, respectively. The procedure is applied for the determination of As in certified reference materials namely wheat flour NIST SRM 1567a (National Institute of Standards and Technology Standard Reference Material), lichen BCR CRM 482 (Institute for Reference Materials, European Commission) and Virginia tobacco leaves CTA-VTL-2 (Poland Academy of Sciences). The results obtained by the present procedure are in good agreement with the certified values and also determined after complete dissolution of samples using closed microwave digestion.
Keywords: Wheat flour; Lichen; Tobacco leaves; Arsenic; Inductively coupled plasma-mass spectrometry; Dry ashing; Microwave digestion;

Computational modeling of the human serum proteome response to colon resection surgery by Cheng-Jian Xu; Huub C.J. Hoefsloot; Martijn Dijkstra; Klaas Havenga; Han Roelofsen; Roel J. Vonk; Age K. Smilde (20-27).
Principal component analysis (PCA) is much used in exploring time-course biological data sets, but does not distinguish variation between time and subjects. This study proposes a new integrated approach by combining analysis of variance (ANOVA) and three component modeling methods. The former was used to separate the between- and within-subject variation, and the latter represent modeling strategies on a scale moving from commonality to individuality. The proposed approach was applied to a surface-enhanced laser desorption and ionization time of flight mass spectrometry (SELDI-TOF-MS) data set of a serum protein expression time course before and after colon resection. Two common biological processes are identified and individual differences among patients were also detected, and the biological relevance of both is discussed.
Keywords: Colon cancer; Modeling; Surface-enhanced laser desorption and ionization time of flight mass spectrometry; Surgery;

Iridium oxide nanoparticles are grown on a glassy carbon electrode by electrodepositing method. The electrochemical behavior and electrocatalytic activity of modified electrode towards reduction of iodate and periodate are studied. The reductions of both ions occur at the unusual positive peak potential of 0.7 V vs. reference electrode. The modified electrode is employed successfully for iodate and periodates detection using cyclic voltammetry, hydrodynamic amperometry and flow injection analysis (FIA). In the performed experiments, flow injection amperometric determination of iodate and periodate yielded calibration curves with the following characteristics: linear dynamic range up to 100 and 80 μM, sensitivity of 140.9 and 150.6 nA μM−1 and detection limits of 5 and 36 nM, respectively. The repeatability of the modified electrode for 21 injections of 1.5 μM of iodate solution is 1.5%. The interference effects of NO2 , NO3 , ClO3 , BrO3 , ClO4 , SO4 2−, Cu2+, Zn2+, Mn2+, Mg2+, Cd2+, Ca2+, Na+, K+, NH4 + and K+, CH3COO and glucose were negligible at the concentration ratio of more than 1000. The obtained attractive analytical performance together with high selectivity and simplicity of the proposed method provide an effective and e novel modified electrode to develop an iodate and periodate sensor. Sensitivity, selectivity, the liner concentration range and the detection limit of the developed sensor are all much better than all known similar sensors in the literature for iodate and periodate determination.
Keywords: Iridium oxide; Nanoparticles; Iodate; Periodate; Flow injection analysis; Amperometry;

Preparation of magnetic strong cation exchange resin for the extraction of melamine from egg samples followed by liquid chromatography–tandem mass spectrometry by Yang Xu; Ligang Chen; Hui Wang; Xiaopan Zhang; Qinglei Zeng; Haoyan Xu; Lei Sun; Qi Zhao; Lan Ding (35-41).
In the work, magnetic strong cation exchange (MSCX) resins were prepared using hydrophobic Fe3O4 magnetite as the magnetically susceptible component, styrene and divinylbenzene as polymeric matrix components, acetyl sulfonate as the sulfonation agent. The resins were successfully applied to the extraction of melamine (MEL) from egg samples. The extraction procedure was carried out in a single step by blending and stirring the sample, extraction solvent and the magnetic resins. The MEL was extracted from the sample matrix then adsorbed onto the resins directly through ion-exchange interaction. When the extraction was completed, the resins with adsorbed analyte were easily separated from the sample matrix by applying an appropriate magnetic field. Main factors affecting the extraction of MEL such as the amount of MSCX resins, extraction time, washing and eluting conditions were optimized. The MEL eluted from the resins was determined by liquid chromatography–tandem mass spectrometry. The linearity of quantification obtained by analyzing matrix-matched standards is in the range of 10–1000 ng g−1. The limit of detection and quantification obtained are 2.6 and 8.8 ng g−1, respectively. The relative standard deviations of intra- and inter-day ranging from 1.6% to 6.5% and from 2.1% to 7.2% are obtained. The recoveries of MEL are in the range of 77.2–99.3%. The proposed method was successfully applied to determine MEL in eggs obtained from different local markets. MEL was detectable with the contents of 43.5 and 234.1 ng g−1 in two samples.
Keywords: Magnetic strong cation exchange resins; Melamine; Egg samples; Liquid chromatography–tandem mass spectrometry;

Monolithic silica spin column extraction and simultaneous derivatization of amphetamines and 3,4-methylenedioxyamphetamines in human urine for gas chromatographic-mass spectrometric detection by Akihiro Nakamoto; Manami Nishida; Takeshi Saito; Izumi Kishiyama; Shota Miyazaki; Katsunori Murakami; Masataka Nagao; Akira Namura (42-46).
A simple, sensitive, and specific method with gas chromatography–mass spectrometry was developed for simultaneous extraction and derivatization of amphetamines (APs) and 3,4-methylenedioxyamphetamines (MDAs) in human urine by using a monolithic silica spin column. All the procedures, such as sample loading, washing, and elution were performed by centrifugation. APs and MDAs in urine were adsorbed on the monolithic silica and derivatized with propyl chloroformate in the column. Methamphetamine-d 5 was used as an internal standard. The linear ranges were 0.01–5.0 μg mL−1 for methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA) and 0.02–5.0 μg mL−1 for amphetamine (AP) and 3,4-methylenedioxyamphetamine (MDA) (coefficient of correlation ≧0.995). The recovery of APs and MDAs in urine was 84–94%, and the relative standard deviation of the intra- and interday reproducibility for urine samples containing 0.1, 1.0, and 4.0 μg mL−1 of APs and MDAs ranged from 1.4% to 13.6%. The lowest detection limit (signal-to-noise ratio ≧ 3) in urine was 5 ng mL−1 for MA and MDMA and 10 ng mL−1 for AP and MDA. The proposed method can be used to perform simultaneous extraction and derivatization on spin columns that have been loaded with a small quantity of solvent by using centrifugation.
Keywords: Monolithic spin column; Propyl chloroformate; Derivatization; Amphetamines; 3,4-Methylenedioxyamphetamines; Gas chromatograph–mass spectrometer;

In order to develop a safety biomarker for atorvastatin, this drug was orally administrated to hyperlipidemic rats, and a metabolomic study was performed. Atorvastatin was given in doses of either 70 mg kg−1  day−1 or 250 mg kg−1  day−1 for a period of 7 days (n  = 4 for each group). To evaluate any abnormal effects of the drug, physiological and plasma biochemical parameters were measured and histopathological tests were carried out. Safety biomarkers were derived by comparing these parameters and using both global and targeted metabolic profiling. Global metabolic profiling was performed using liquid chromatography/time of flight/mass spectrometry (LC/TOF/MS) with multivariate data analysis. Several safety biomarker candidates that included various steroids and amino acids were discovered as a result of global metabolic profiling, and they were also confirmed by targeted metabolic profiling using gas chromatography/mass spectrometry (GC/MS) and capillary electrophoresis/mass spectrometry (CE/MS). Serum biochemical and histopathological tests were used to detect abnormal drug reactions in the liver after repeating oral administration of atorvastatin. The metabolic differences between control and the drug-treated groups were compared using PLS-DA score plots. These results were compared with the physiological and plasma biochemical parameters and the results of a histopathological test. Estrone, cortisone, proline, cystine, 3-ureidopropionic acid and histidine were proposed as potential safety biomarkers related with the liver toxicity of atorvastatin. These results indicate that the combined application of global and targeted metabolic profiling could be a useful tool for the discovery of drug safety biomarkers.
Keywords: Atorvastatin; Metabolomics; Biomarker; Mass spectrometry;

A pair of pseudo-enantiomers, tertiary amine appended trans-4-hydroxyproline derivatives were designed, synthesized, and evaluated as chiral selectors for enantiomer analysis of DNB-amino acid and their amides, in single-stage electrospray ionization/mass spectrometric experiments. The chiral selectors were designed to remove the interaction of the hydroxyl group of trans-4-hydroxyproline as well as separate the ionization site from the sites required for effective chiral recognition. Addition of a chiral analyte to a solution containing two pseudo-enantiomeric chiral selectors, affords selector–analyte complexes in the electrospray ionization mass spectrum where the ratio of these complexes is dependent on the enantiomeric composition of the analyte. The relationship between the ratio of the selector–analyte complexes in the electrospray ionization mass spectrum and the enantiomeric composition of the analyte can be used to relate the extent of the measured enantioselectivity and for quantitative enantiomeric composition determinations. Effects of acid modifiers (ammonium chloride, acetic acid, formic acid and hydrochloric acid) and instrument conditions on the selector–analyte ion intensity and the enantioselectivity (α MS) were investigated. The largest α MS was observed using ammonium chloride at a concentration around 0.5–1 mM at desolvation temperature of 150 °C. Capillary voltage has little effects on α MS values. The sense of chiral recognition by MS is consistent with what is observed chromatographically. Quantitative enantiomeric composition determinations for N-(3,5-dinitrobenzoyl) leucinyl butylamide were performed. A comparison to the enantioselectivities towards a scope of analytes observed by chiral HPLC using a 3,5-dimethylanilide-proline-derived chiral stationary phase, is presented.
Keywords: Chiral recognition; Electrospray ionization-mass spectrometry; Tertiary amine appended trans-4-hydroxyproline derivatives; Gas phase;

In vitro methylation by methanol: Proteomic screening and prevalence investigation by Guoqiang Chen; Hui Liu; Xiaodong Wang; Zhili Li (67-75).
It is assumed that much more functional importance for protein activity than expected may be granted by methylation that occurs at the side-chain of aspartate or glutamate residue. In vitro methylation mainly comes from the use of methanol in sample preparation prior to MS analysis. In this study, we first performed the methylation site-directed proteomic screening of bovine serum albumin, ovalbumin and 20S proteasome for gel staining using a meaningfully indicative MS-pattern of peak tag (termed as 4P tag) and manual inspection for mass spectral data. As a result, there were 17 proteolytic peptides with 20 modified sites confirmed to be in vitro methylated. Subsequently, the prevalence investigation was performed, focusing on the reaction kinetic behavior of in vitro methylation. This study provided a simple and robust approach for confirmation of in vitro methylation by methanol, as well as the precautious guide for the use of methanol in proteomic study.
Keywords: In vitro methylation; Matrix/sample preparation; Methanol; Proteasome; Proteomic screening;

The development of an efficient analytical protocol for the reliable identification of the biosynthetic intermediates found in microbial cultures, which usually produce complex intermediates of the metabolites of interest, is both challenging and essential for further studies into gene-to-metabolite networks. A simple and highly selective method for detecting the biosynthetic intermediates involved in the aminoglycosidic nebramycin pathway of Streptomyces tenebrarius was developed and validated. Cleanup utilizing a solid-phase extraction (OASIS MCX SPE) technique provides a simple and reproducible method for extracting the nebramycin factors from a fermentation broth. The separation of each factor through a reversed-phase C18 column using an ion-pairing reagent allowed the simultaneous profiling of the aminoglycosides. By employing the authentic tobramycin spiked into a blank fermentation medium, the combined use of acid extraction, OASIS MCX SPE cleanup, and HFBA (heptafluorobutyric acid)-mediated chromatographic separation coupled with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) detection was proven to be sufficiently accurate and reliable to analyze the nebramycin factors produced in a culture broth. The detection limit of tobramycin spiked in the culture broth was approximately 1.8 ng mL−1. The mean recovery ranged from 89 to 92%, the intra- and inter-day precision (RSD) was <6% and their accuracy ranged from 88 to 93%. A total of nine nebramycin factors including apramycin, 6″-O-carbamoylkanamycin B, 6″-O-carbamoyltobramycin, 3′-hydoxyapramycin, tobramycin, kanamycin B, NK-1012-1, nebramine, and neamine were identified. This is the first report on the integrated LC-ESI-MS/MS characterization of a wide range of nebramycin factors from a bacterial fermentation broth.
Keywords: Nebramycin factor; Streptomyces tenebrarius; Liquid chromatography-tandem mass spectrometry; Aminoglycoside; Fermentation;

A new, simple and highly sensitive method for spectrofluorimetric determination of amiloride (AMI) and furosemide (FUR) in pharmaceuticals is presented. The proposed method is based on the separation of AMI from FUR by solid-phase extraction using a nylon membrane, followed by spectrofluorimetric determination of both drugs, on the solid surface and the filtered aqueous solution, respectively. AMI shows low native fluorescence, but its separation-preconcentration by immobilization (solid-phase extraction) on nylon membrane surface provides a considerable enhancement in fluorescence intensity. The fluorescence determination is carried out at λ ex  = 237, λ em  = 415 nm for FUR; and λ ex  = 365, λ em  = 406 nm for AMI. The calibration graphs are linear in the range 3.20 × 10−4 to 0.8 μg mL−1and 1.33 × 10−3 to 4.0 μg mL−1, for AMI and FUR, respectively, with a detection limit of 9.62 × 10−5 and 4.01 × 10−4  μg mL−1 (S/N  = 3). The commonly found excipients in commercial pharmaceutical formulations do not interfere. The developed method is successfully applied to the determination of both drugs in pharmaceutical formulations.
Keywords: Amiloride; Furosemide; Pharmaceutical formulation; Spectrofluorimetry; Solid-phase extraction; Nylon membrane;

Photoelectrochemical determination of inorganic mercury in aqueous solutions by Jessica Chamier; Joy Leaner; Andrew M. Crouch (91-96).
An analytical method using an optical probe in a photoelectrochemical cell for the sensitive and selective determination of aqueous Hg2+ is presented. A previously synthesized Hg2+ selective chemosensor, proven to be Hg2+ sensitive up to 2 μg L−1, has been immobilized onto indium tin oxide (ITO) electrodes in a composite form with polyaniline. The coated ITO electrode was placed in a photoelectrochemical cell under closed circuit conditions in which the optical recognition of the chemosensor was converted to a measurable signal. A composite of the fluorescent chemosensor, Rhodamine 6G derivative (RS), and polyaniline (PANI) was immobilized on ITO glass plates and subjected to photovoltage measurements in the absence and presence of Hg2+. The optical responses of the coated electrode were used to determine the sensitivity and selectivity of the immobilized sensor to Hg2+ in the presence of background ions. The optical response of the PANI-dye coated electrode increased linearly with increasing Hg2+ concentration in the range 10–150 μg L−1, with a detection limit of 6 μg L−1.
Keywords: Photoelectrochemical detection; Inorganic mercury; Chemosensor; Polyaniline; Rhodamine 6G;

Novel colorimetric sensor for oral malodour by Nethaji Alagirisamy; Sarita S. Hardas; Sujatha Jayaraman (97-102).
Volatile sulphur compounds are the primary constituents of oral malodour. Quantitative tools for the detection of oral malodour are beneficial to evaluate the intensity of malodour, analyse its causes and monitor the effectiveness of customized treatments. We have developed an objective, cost effective, do-it-yourself colorimetric sensor for oral malodour quantification. The sensor consisted of a sensing solution, a gas sampling unit for collecting a known volume of mouth air and a photometric detector. The sensing solution was iodine and the depletion of iodine on reaction with hydrogen sulphide was detected colorimetrically using starch. The detection limit of the sensor is 0.05 μg L−1 of hydrogen sulphide, which is fit-for-purpose for oral malodour detection in healthy subjects as well as halitosis patients. Volatile sulphur compounds in mouth air were quantified in healthy human volunteers using this portable sensor and the detected levels were in the range of 0.2–0.4 μg L−1. There was a good correlation between the VSC levels detected by the colorimetric sensor and halimeter (R 2  = 0.934). The developed sensor can be easily fabricated in the laboratory, and it shows high potential to be used as a clinical evaluation tool for oral malodour assessments.
Keywords: Oral malodour; Volatile sulphur compounds; Hydrogen sulphide; Colorimetry; Portable sensor; Iodine–starch sensor;

Preliminary studies of mixed films composed of oligonucleotides and poly(2-hydroxyethyl methacrylate) (PHEMA) have recently been shown to enhance the selectivity for detection of 3 base-pair mismatched (3 bpm) oligonucleotide targets. Evaluation of selectivity for detection of single nucleotide polymorphisms (SNP) using such mixed films has now been completed. The selectivity was quantitatively determined by considering the sharpness of melt curves and melting temperature differences (ΔT m) for fully complementary targets and SNPs. Stringency conditions were investigated, and it was determined that the selectivity was maximized when a moderate ionic strength was used (0.1–0.6 M). Increases of ΔT m when using mixed films were up to 3-fold larger compared to surfaces containing only immobilized oligonucleotide probes. Concurrently, increases in sharpness of melt curves for 1 bpm targets were observed to be up to 2-fold greater for mixed films. The co-immobilization of PHEMA resulted in a more homogeneous distribution of oligonucleotide probes on surfaces. Lifetime measurements of fluorescence emission from immobilized oligonucleotide probes labeled with Cy3 dye indicated the difference in microenvironment of immobilized oligonucleotides in the presence of PHEMA.
Keywords: Single nucleotide polymorphism; Melt curve; Oligonucleotides; Glass; Cy3; Fluorescence lifetime; Mixed film; Poly(2-hydroxyethyl methacrylate);

Nanostructured biochip for label-free and real-time optical detection of polymerase chain reaction by Ha Minh Hiep; Kagan Kerman; Tatsuro Endo; Masato Saito; Eiichi Tamiya (111-116).
In this report, Au-coated nanostructured biochip with functionalized thiolated primers on its surface is developed for label-free and real-time optical detection of polymerase chain reaction (PCR). A PCR chamber of 150 μm in thickness containing Au-coated nanostructured substrate in the bottom layer was bordered with SU-8 100 walls. After immobilization of 5′-thiolated primers on the surface, simultaneous DNA amplification and detection were performed without any labeled molecules via the relative reflected intensity (RRI) of Au-coated nanostructured substrate. When human genomic DNA at several concentrations of 0.2, 0.5 and 1 ng μL−1 was included in the initial DNA samples, the increases in the RRI peak values were clearly observed with the increasing PCR cycle numbers. We found that the starting point of the optical signal, which was divergent from the background in our PCR biochip, was around 3–4 cycles, much lower than that of the fluorescent real-time PCR analysis (around 23–25 cycles). Our proposed PCR device using Au-coated nanostructured substrate holds noteworthy promise for rapid, label-free and real-time DNA detection for point-of-care testing (POCT) applications.
Keywords: Real-time polymerase chain reaction; Label-free optical detection of polymerase chain reaction; Au-coated nanostructured biochip;

Integrated explosive preconcentrator and electrochemical detection system for 2,4,6-trinitrotoluene (TNT) vapor by Karel Cizek; Chad Prior; Chongdee Thammakhet; Michal Galik; Kevin Linker; Ray Tsui; Avi Cagan; John Wake; Jeff La Belle; Joseph Wang (117-121).
This article reports on an integrated explosive-preconcentration/electrochemical detection system for 2,4,6-trinitrotoluene (TNT) vapor. The challenges involved in such system integration are discussed. A hydrogel-coated screen-printed electrode is used for the detection of the thermally desorbed TNT from a preconcentration device using rapid square wave voltammetry. Optimization of the preconcentration system for desorption of TNT and subsequent electrochemical detection was conducted yielding a desorption temperature of 120 °C under a flow rate of 500 mL min−1. Such conditions resulted in a characteristic electrochemical signal for TNT representing the multi-step reduction process. Quantitative measurements produced a linear signal dependence on TNT quantity exposed to the preconcentrator from 0.25 to 10 μg. Finally, the integrated device was successfully demonstrated using a sample of solid TNT located upstream of the preconcentrator.
Keywords: Trinitrotoluene; Vapor detection; Screen-printed electrodes; Explosive detection;

Use of 3-(4-hydroxyphenyl)propionic acid as electron donating compound in a potentiometric aflatoxin M1-immunosensor by Steffen Rameil; Peter Schubert; Peter Grundmann; Richard Dietrich; Erwin Märtlbauer (122-127).
We developed a potentiometric aflatoxin M1-immunosensor which utilizes 3-(4-hydroxyphenyl)propionic acid (p-HPPA) as electron donating compound for horseradish peroxidase (HRP; EC The assay system consists of a polypyrrole-surface-working electrode coated with a polyclonal anti-M1 antibody (pAb–AFM1), a Ag/AgCl reference electrode and a HRP–aflatoxin B1 conjugate (HRP–AFB1 conjugate).To optimize the potentiometric measuring system p-HPPA as well as related compounds serving as electron donating compounds were compared. Also the influence of different buffer systems, varying pH and substrate concentrations on signal intensity was investigated. Our results suggest that reaction conditions that favor the formation of Pummerer's type ketones lead to an increase in signal intensity rather than formation of fluorescent dye. Comparison with commercial ready-to-use HRP electron donating compounds such as 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), o-phenylenediamine (OPD) or 3,3′,5,5′-tetramethylbenzidine (TMB) showed that only 34%, 77% and 49% of the signal intensity of p-HPPA were reached, respectively.The optimized assay had a detection limit of 40 pg mL−1 and allowed detection of 500 pg mL−1 (FDA action limit) aflatoxin M1 (AFM1) in pasteurized milk and UHT-milk containing 0.3–3.8% fat within 10 min without any sample treatment. The working range was between 250 and 2000 pg mL−1 AFM1.
Keywords: 3-(4-Hydroxyphenyl)propionic acid; Aflatoxin M1; Milk; Pummerer's ketone; Potentiometric immunosensor;