Analytical and Bioanalytical Chemistry (v.402, #6)

is a professor teaching chemistry at the University of São Paulo. Dr. Correia’s background on atomic absorption spectrometry allows connecting his current research interests on instructional science and the challenges of teaching analytical chemistry. The use of concept mapping for knowledge and information management has driven the activities of the Cmapping research group at the School of Arts, Sciences and Humanities (for more information, see http://www.each.usp.br/cmapping/ ). The research projects under development aim to develop instructional materials to training beginners to become proficient map makers.

started her professional life as a chemist, following her first degree in Chemical and Analytical Science with a PhD in Physical Chemistry. After a brief period in post-doctoral research, she discovered an interest in transferable skills whilst teaching undergraduate chemists communication skills. After training as a university careers adviser, in 2000, she founded a company ( www.shintonconsulting.com ) and now delivers career guidance and skills training to academic researchers and scientists across the UK and Europe. Find her on Twitter as @sarashinton.

Comprehensive two-dimensional gas chromatography in metabolomics by Martin F. Almstetter; Peter J. Oefner; Katja Dettmer (1993-2013).
One of the major objectives in metabolomics is the identification of subtle changes in metabolite profiles as affected by genetic or environmental factors. Comprehensive two-dimensional gas chromatography (GC × GC) hyphenated to a fast-acquisition mass spectrometer is a well-established analytical technique to study the composition of complex samples due to its enhanced separation capacity, sensitivity, peak resolution, and reproducibility. This review reports applications of GC × GC to metabolomics studies of sample of different types (biofluid, cells, tissue, bacteria, yeast, plants), and discusses its advantages and limitations.
Keywords: Metabolomics; Metabolite profiling; Metabolic fingerprinting; Comprehensive two-dimensional gas chromatography

Infrared spectroscopic analysis of mononuclear leukocytes in peripheral blood from Alzheimer’s disease patients by Pedro Carmona; Marina Molina; Miguel Calero; Félix Bermejo-Pareja; Pablo Martínez-Martín; Isabel Alvarez; Adolfo Toledano (2015-2021).
Peripheral mononuclear leukocytes from Alzheimer’s disease (AD) patients were analyzed by infrared spectroscopy and their spectroscopic properties were compared with those from age-matched healthy controls. Two-dimensional correlation analysis of mean spectra measured at various disease stages shows that the protein secondary structure from AD patients involves β-sheet enrichment and carbonyl intensity increase relative to healthy controls. The area percentages of β-sheets, which were obtained by using a peak ratio second-derivative spectral treatment, were used for receiver operating characteristic (ROC) analysis to distinguish between patients with AD and age-matched healthy controls. The critical concentration and area under the curve (AUC) were determined by this curve analysis which showed a good performance for this quantitative assay. The results were 90% sensitivity and 90.5% specificity for determinations involving mild and moderate AD patients, and 82.1% sensitivity and 90.5% specificity for determinations involving patients at the three AD stages (mild, moderate, and severe). The AUC was greater than 0.85 in both scenarios. Taken together these results show that healthy controls are distinguished from mild and moderate AD patients better than from patients with severe disease and suggest that this infrared analysis is a promising strategy for AD diagnostics.
Keywords: Infrared spectroscopy; Mononuclear leukocytes; Alzheimer’s disease; Diagnostics

Phytosterol oxidation products (POPs) have been suggested to exert adverse biological effects similar to, although less severe than, their cholesterol counterparts. For that reason, their analysis in human plasma is highly relevant. Comprehensive two-dimensional gas chromatography (GC×GC) coupled with time-of-flight mass spectrometry (TOF-MS) has been proven to be an extremely powerful separation technique for the analysis of very low levels of target compounds in complex mixtures including human plasma. Thus, a GC×GC/TOF-MS method was developed and successfully validated for the simultaneous quantification of ten POPs in human plasma. The calibration curves for each compound showed correlation coefficients (R 2) better than 0.99. The detection limits were below 0.1 ng mL−1. The recovery data were between 71.0% and 98.6% (RSDs <10% for all compounds validated). Good results were obtained for within- and between-day repeatability, with most values being below 10%. In addition, non-targeted sterol metabolites were also identified with the method. The concentrations of POPs found in human plasma in the current study are between 0.3 and 4.5 ng mL−1, i.e., 10–100 times lower than the typical values found for cholesterol oxidation products. Figure Extracted regions of GC×GC/TOF-MS chromatograms displaying derivatives of sterol oxidation products and sterols for plasma samples
Keywords: Comprehensive two-dimensional gas chromatography; GC×GC; Phytosterols; Plant sterols; Phytosterol oxidation products; Human plasma

A new methodology for simultaneous quantification of total-Aβ, Aβx-38, Aβx-40, and Aβx-42 by column-switching LC/MS/MS by Ken-ichi Watanabe; Chihiro Ishikawa; Hiroshi Kuwahara; Kimihiko Sato; Setsuko Komuro; Tetsuya Nakagawa; Naruaki Nomura; Shiro Watanabe; Masashi Yabuki (2033-2042).
This article details the development of a novel method that overcomes the drawbacks of sandwich ELISA (sELISA) and allows reliable evaluation of simultaneous quantification of the amyloid (Aβ)-peptides, total-Aβ, Aβx-38, Aβx-40, and Aβx-42, in rat brain by optimized sample purification and column-switching liquid chromatographic-tandem mass spectrometry (LC/MS/MS). This method provides accurate analyses of total-Aβ, Aβx-38, Aβx-40, and Aβx-42 with a linear calibration range between 0.05 and 45 ng/mL. Verification for accuracy and precision of biological samples were determined by a standard addition and recovery test, spiked with synthetic Aβ1-38, Aβ1-40, and Aβ1-42 into the rat brain homogenate. This method showed <20% relative error and relative standard deviation, indicating high reproducibility and reliability. The brain concentrations of total-Aβ, Aβx-38, Aβx-40, and Aβx-42 after oral administration of flurbiprofen in rats were measured by this method. Aβx-42 concentrations (4.57 ± 0.69 ng/g) in rats administered flurbiprofen were lower than those in untreated rats (6.48 ± 0.93 ng/g). This was consistent with several reports demonstrating that NSAIDs reduced the generation of Aβ. We report here a method that allows not only the quantification of specific molecular species of Aβ but also simultaneous quantification of total-Aβ, Aβx-38, Aβx-40, and Aβx-42, thus overcoming the drawbacks of sELISA.
Keywords: Total-Aβ, Aβx-38, Aβx-40, and Aβx-42; Biomarker; Rat brain; Quantification; Column-switching LC/MS/MS

A novel stationary phase prepared by the thermal immobilization of poly(dimethylsiloxane) onto the surface of silica (PDMS–SiO2) has been described, evaluated and compared with 229 commercially available RP-LC stationary phases using the Tanaka column classification protocol. The phase exhibited many unique chromatographic properties and, based on the phases in the database, was most similar to the fluoroalkylated phases (aside from the obvious lack of fluoro selectivity imposed by the C–F dipole). The phase exhibited classic reversed-phase behaviour in acid mobile phase conditions and mixed-mode reversed-phase/cation-exchange retention behaviour in neutral mobile phase conditions. The phase exhibited acceptable stability at both low and intermediate pH, conditions which should impart optimum chromatographic selectivity to the phase. Retention of basic analytes was shown to occur by a “three site model” as proposed by Neue. This new PDMS–SiO2 stationary phase is extremely interesting in that the dominancy of its hydrophobic and ion-exchange interactions can be controlled by the influence of mobile phase pH, buffer type and concentration. The PDMS–SiO2 stationary phase may provide a complementary tool to reversed-phase and HILIC stationary phases. The present results highlight the fact that the type of buffer, its concentration and pH can not only affect peak shape but also retention, selectivity and hence chromatographic resolution. Therefore, in method development and optimization strategies it is suggested that more emphasis should be given to the evaluation of these mobile phase operating parameters especially when basic solutes are involved.
Keywords: Mixed-mode stationary phase; Reversed-phase/cation-exchange stationary phase; Immobilized polysiloxane; Chromatographic test; Chemometrics; Basic solutes

Multiplexed quantification of proteins adsorbed to surface-modified and non-modified microdialysis membranes by Andreas P. Dahlin; Klas Hjort; Lars Hillered; Marcus O. D. Sjödin; Jonas Bergquist; Magnus Wetterhall (2057-2067).
A simple and straightforward method for discovery and quantification of proteins adsorbed onto delicate and sensitive membrane surfaces is presented. The adsorbed proteins were enzymatically cleaved while still adsorbed onto the membranes using an on-surface enzymatic digestion (oSED). This was followed by isobaric tagging, nanoliquid chromatography, and tandem mass spectrometry. Protein adsorption on tri-block copolymer Poloxamer 407 surface-modified microdialysis (MD) membranes were compared with protein adsorption on unmodified MD membranes. Ventricular cerebrospinal fluid (vCSF) kept at 37 °C was used as sample matrix. In total, 19 proteins were quantified in two biological replicates. The surface-modified membranes adsorbed 33% less proteins than control membranes and the most abundant proteins were subunits of hemoglobin and clusterin. The adsorption of clusterin on the modified membranes was on average 36% compared to control membranes. The most common protein in vCSF, Albumin, was not identified adsorbed to the surface at all. It was also experimentally verified that oSED, in conjunction with tandem mass spectrometry can be used to quantify femtomole amounts of proteins adsorbed on limited and delicate surfaces, such as MD membranes. The method has great potential and can be used to study much more complex protein adsorption systems than previously reported. Figure On-surface enzymatic digestion (oSED) of proteins adsorbed onto microdialysis membranes. The adsorbed proteins are enzymatically cleaved while still adsorbed onto the membranes. The released tryptic peptides are then analysed by isobaric tagging, nanoliquid chromatography, and tandem mass spectrometry
Keywords: Microdialysis; On-surface enzymatic digestion (oSED); Mass spectrometry (MS); Cerebrospinal fluid (CSF); Interface/surface analysis; Tri-block copolymer surface modification

Characterization of different FAD-dependent glucose dehydrogenases for possible use in glucose-based biosensors and biofuel cells by Muhammad Nadeem Zafar; Najat Beden; Dónal Leech; Christoph Sygmund; Roland Ludwig; Lo Gorton (2069-2077).
In this study, different flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) were characterized electrochemically after “wiring” them with an osmium redox polymer [Os(4,4′-dimethyl-2,2′-bipyridine)2(PVI)10Cl]+ on graphite electrodes. One tested FADGDH was that recently discovered in Glomerella cingulata (GcGDH), another was the recombinant form expressed in Pichia pastoris (rGcGDH), and the third was a commercially available glycosylated enzyme from Aspergillus sp. (AspGDH). The performance of the Os-polymer “wired” GDHs on graphite electrodes was tested with glucose as the substrate. Optimal operational conditions and analytical characteristics like sensitivity, linear ranges and current density of the different FADGDHs were determined. The performance of all three types of FADGDHs was studied at physiological conditions (pH 7.4). The current densities measured at a 20 mM glucose concentration were 494 ± 17, 370 ± 24, and 389 ± 19 μA cm−2 for GcGDH, rGcGDH, and AspGDH, respectively. The sensitivities towards glucose were 2.16, 1.90, and 1.42 μA mM−1 for GcGDH, rGcGDH, and AspGDH, respectively. Additionally, deglycosylated rGcGDH (dgrGcGDH) was investigated to see whether the reduced glycosylation would have an effect, e.g., a higher current density, which was indeed found. GcGDH/Os-polymer modified electrodes were also used and investigated for their selectivity for a number of different sugars. Figure Comparison of different parameters for GDHs/Os-polymer modified electrodes
Keywords: Glucose biosensor; FAD; Glucose dehydrogenase; Os-polymer; Deglycosylation; Biofuel cell

Lambda genomic DNA quantification using ultrasonic treatment followed by liquid chromatography–isotope dilution mass spectrometry by Lianhua Dong; Chao Zang; Jing Wang; Liang Li; Yunhua Gao; Liqing Wu; Peng Li (2079-2088).
Quantification of genomic DNA that is traceable to the SI was performed successfully by measuring the individual nucleotides. Specifically, ultrasound was used to shear lambda genomic DNA into fragments of less than 200 base pairs, followed by deoxyribonuclease Ι and phosphodiesterase Ι digestion and liquid chromatography–isotope dilution mass spectrometry (LC-IDMS) quantification to estimate the mass fraction of the lambda DNA, based on the constituent deoxynucleotide monophosphates (dNMPs) within the molecule. Digital PCR (dPCR) was employed to quantify the same lambda DNA solution to provide independent data for comparing the performance of two quantitative methods. On the basis of the LC-IDMS measurement after ultrasonic treatment of the sample, the concentration of lambda DNA was 273.1 ± 9.8 μg/g (expanded uncertainty at the 95% confidence interval). This shows good agreement with the data from dPCR. Additionally, the result calculated on the basis of the sum of the concentrations of the four dNMPs is the same as that calculated on the basis of the sequence, which indicates that knowledge of the DNA sequence and length is unnecessary to measure the total DNA concentration when applying ultrasonic treatment–LC-IDMS.
Keywords: Isotope dilution mass spectrometry; DNA quantification; Digital PCR; Ultrasonic

Beta endorphin (β-END) is recognised as one of the most significant endogenous neuropeptides, responsible for a wide range of biological activities in the body. However, within the body β-END is exposed to hydrolysis by a variety of enzymes. In this study, we investigated the metabolism and fragmentation pattern of β-END in rat inflamed tissue, in rat serum and in trypsin solution. β-END (1-31)-rat was incubated at 37 °C in each matrix for different incubation times. The resultant fragments were separated using a C4 column and detected by mass spectrometry using total ion current mode. Structural information for the fragments was elucidated using tandem mass spectrometry. Incubation of β-END (1-31)-rat in trypsin solution and in rat serum resulted in 8 and 13 fragments, respectively. Incubation in inflamed rat paw tissue resulted in 22 fragments at pH 7.4 and 26 fragments at pH 5.5. Some of these fragments were common to both pH values. The degradation of β-END (1-31)-rat in inflamed tissue at pH 5.5 was faster than that at pH 7.4. Secondary fragmentation of some larger primary fragments was also observed in this study.
Keywords: Beta endorphin; Metabolism; Fragments; Inflamed tissue; LC-MS-MS

A lectin-coupled, multiple reaction monitoring based quantitative analysis of human plasma glycoproteins by mass spectrometry by Yeong Hee Ahn; Park Min Shin; Eun Sun Ji; Hoguen Kim; Jong Shin Yoo (2101-2112).
Aberrant protein glycosylation may be closely associated with cancer pathology. To measure the abundance of protein glycoforms with a specific glycan structure in plasma samples, we developed a lectin-coupled multiple reaction monitoring (MRM)-based mass spectrometric method. It was confirmed that the method could provide reproducible results with precision sufficient to distinguish differences in the abundance of protein glycoforms between individuals. Plasma samples prepared from hepatocellular carcinoma (HCC) patients without immuno-depletion of highly abundant plasma proteins were fractionated by use of fucose-specific aleuria aurantia lectin (AAL) immobilized on magnetic beads by use of a biotin–streptavidin conjugate. The lectin-captured fractions were digested by trypsin and profiled by tandem mass spectrometry. From the proteomic profiling data, target glycoproteins were selected and analyzed quantitatively by MRM-based analysis. The reproducibility of MRM-based quantification of the selected target proteins was reliable, with precision (CV; ≤14% for batch-to-batch replicates and ≤19% for replicates over three days) sufficient to distinguish differences in the abundance of AAL-captured glycoforms between individual plasma samples. This lectin-coupled, MRM-based method, measuring only lectin-captured glycoforms of a target protein rather than total target protein, is a tool for monitoring differences between individuals by measuring the abundance of aberrant glycoforms of a target protein related to a disease. This method may be further applied to rapid verification of biomarker candidates involved in aberrant protein glycosylation in human plasma. Figure A lectin-coupled, MRM-based approach for quantitative analysis of plasma glycoproteins
Keywords: Plasma; Glycoprotein; Lectin; Multiple reaction monitoring; Mass spectrometry; Cancer

Acrylonitrile (AN), a widely used industrial chemical also found in tobacco smoke, has been classified as a possible human carcinogen (group 2B) by the International Agency for Research on Cancer. AN can be detoxified by glutathione S-transferase (GST) to form glutathione (GSH) conjugates in vivo. It can be metabolically activated by cytochrome P450 2E1 to form 2-cyanoethylene oxide, which can also be detoxified by GST to generate GSH conjugates. The GSH conjugates can be further metabolized to mercapturic acids (MAs), namely, N-acetyl-S-(2-cyanoethyl)cysteine (CEMA), N-acetyl-S-(2-hydroxyethyl)cysteine (HEMA), and N-acetyl-S-(1-cyano-2-hydroxyethyl)cysteine (CHEMA). This study developed an ultraperformance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method to quantitatively profile the major AN urinary metabolites (CEMA, HEMA, and CHEMA) to assess AN exposure, as well as analyze urinary cotinine (COT) as an indicator for tobacco smoke exposure. The limits of quantitation were 0.1, 0.1, 1.0, and 0.05 μg/L for HEMA, CEMA, CHEMA, and COT, respectively. This method was applied to analyze the three AN-derived MAs in 36 volunteers with no prior occupational AN exposure. Data analysis showed significant correlations between the level of COT and the levels of these MAs, suggesting them as biomarkers for exposure to low levels of AN. The results demonstrate that a highly specific and sensitive UPLC-MS/MS method has been successfully developed to quantitatively profile the major urinary metabolites of AN in humans to assess low AN exposure. Figure Simultaneous quantitation of 3 acrylonitrile-derived mercapturic acids and cotinine in human urine samples with on-line solid-phase extraction LC-MS/MS.
Keywords: Acrylonitrile; Ultraperformance liquid chromatography–tandem mass spectrometry; Cotinine; Mercapturic acids

Quadrupole-based inductively coupled–mass spectrometry (ICP-MS) with pneumatic nebulization as a means of sample introduction was employed for quantification of platinum in blood and tissue samples of rats with peritoneal carcinomatosis, receiving intraperitoneal treatment with the Pt-containing chemotherapeutic drug oxaliplatin, and in the perfusate solution used for this purpose. The Pt levels were measured for various treatment conditions, i.e., with and without supporting treatment with the drug bevacizumab and at two different temperatures. Limits of detection obtained for platinum in blood and tissue samples were 0.3 and 2.0 pg g , −1 respectively. Evaluation of drug penetration into the tumor, under different conditions of treatment, was carried out via laser ablation–ICP-MS. Quantitative mapping of the Pt distribution in tissue sections of rat was attempted relying on gelatin standards. The results show an influence of the temperature at which the treatment is carried out, while supporting administration of the drug bevacizumab did not seem to affect the results.
Keywords: Platinum; Laser ablation–ICP–mass spectrometry; Biological samples; Quantitative mapping; Chemotherapeutic drugs

A capillary electrophoresis-inductively coupled plasma mass spectrometry (CE-ICP-MS) method was developed for separation of the free oxaliplatin drug substance from liposome-entrapped oxaliplatin. Simultaneous determination of phosphorous and platinum opened the possibility to simultaneously monitor the liposomes (phospholipids) and platinum-based drug. In order to suppress the interferences, argon gas was used as a collision gas in ICP-MS. A detection limit of 29 ng/mL of platinum and a precision of 2.9% (for 10 μg/mL of oxaliplatin standard) were obtained. Measurement of the total concentration of free and encapsulated oxaliplatin by CE-ICP-MS was compared with total determination by ICP-MS after microwave digestion and showed a good agreement. A liposomal formulation of oxaliplatin based on PEGylated liposomes was used as a model drug formulation. Studies of accelerated drug release induced by sonication and phospholipase A2 catalyzed hydrolysis were performed. It was demonstrated that the CE-ICP-MS was an efficient in vitro characterization method in the development and quality assurance purposes of lipsome-based formulation of metallodrugs. Figure Free and liposome-encapsulated platinum drug is distinguished by simultaneous detection of phosphorous and platinum by CE-ICP-MS
Keywords: CE-ICP-MS; Capillary electrophoresis; PEGylated liposomes; Oxaliplatin; Phospholipase A2 ; Sonication

Human liver microsomes (HLMs) are used to simulate human xenobiotic metabolism in vitro. In forensic and clinical toxicology, HLMs are popularly used to study the metabolism of new designer drugs for example. In this work, we present an automated online extraction system we developed for HLM experiments, which was compared to a classical offline approach. Furthermore, we present studies on the metabolism of 11 cathinones; for eight of these, the metabolism has not previously been reported. Metabolites were identified based on MS2 and MS3 scans. Fifty-three substances encompassing various classes of drugs were employed to compare the established offline and the new online methods. The metabolism of each of the following 11 cathinones was studied using the new method: 3,4-methylenedioxy-N-benzylcathinone, benzedrone, butylone, dimethylcathinone, ethylone, flephedrone, methedrone, methylone, methylethylcathinone, naphyrone, and pentylone. The agreement between the offline and the online methods was good; a total of 158 metabolites were identified. Using only the offline method, 156 (98.7%) metabolites were identified, while 151 (95.6%) were identified using only the online method. The metabolic pathways identified for the 11 cathinones included the reduction of the keto group, desalkylation, hydroxylation, and desmethylenation in cathinones containing a methylenedioxy moiety. Our method provides a straightforward approach to identifying metabolites which can then be added to the library utilized by our clinical toxicological screening method. The performance of our method compares well with that of an established offline HLM procedure, but is as automated as possible.
Keywords: LC-MS; Human liver microsomes; Cathinones; Online extraction; Human metabolism

Aptasensor for ampicillin using gold nanoparticle based dual fluorescence–colorimetric methods by Kyung-Mi Song; Euiyoung Jeong; Weejeong Jeon; Minseon Cho; Changill Ban (2153-2161).
A gold nanoparticle based dual fluorescence–colorimetric method was developed as an aptasensor to detect ampicillin using its single-stranded DNA (ssDNA) aptamer, which was discovered by a magnetic bead-based SELEX technique. The selected aptamers, AMP4 (5′-CACGGCATGGTGGGCGTCGTG-3′), AMP17 (5′-GCGGGCGGTTGTATAGCGG-3′), and AMP18 (5′-TTAGTTGGGGTTCAGTTGG-3′), were confirmed to have high sensitivity and specificity to ampicillin (K d, AMP7 = 9.4 nM, AMP17 = 13.4 nM, and AMP18 = 9.8 nM, respectively). The 5′-fluorescein amidite (FAM)-modified aptamer was used as a dual probe for observing fluorescence differences and color changes simultaneously. The lower limits of detection for this dual method were a 2 ng/mL by fluorescence and a 10 ng/mL by colorimetry for ampicillin in the milk as well as in distilled water. Because these detection limits were below the maximum residue limit of ampicillin, this aptasensor was sensitive enough to detect antibiotics in food products, such as milk and animal tissues. In addition, this dual aptasensor will be a more accurate method for antibiotics in food products as it concurrently uses two detection methods: fluorescence and colorimetry. Figure The dual fluorescence-colorimetric method for the detection of ampicillin using the AMP17 ssDNA aptamer. The FAM-AMP17 aptamer is quenched by adsorption onto the surface of the AuNPs. If ampicillin and salt (NaCl) are added to the solution, fluorescence is recovered and the AuNPs aggregate. Conversely, the AuNP solution treated with only salt shows no obvious changes.
Keywords: Ampicillin; ssDNA aptamer; Gold nanoparticle; Fluorescence; Colorimetry; Aptasensor

On the basis of the resonance light scattering (RLS) of Ag nanoparticles (AgNPs), an RLS off–on system was developed for studies of the selective interaction between adriamycin (ADM) and DNA. In this strategy, addition of ADM could induce a proportional decrease in the RLS intensity of AgNPs; this could be used to detect trace amounts of ADM with a detection limit of 12.75 ng mL−1 in the range 0.021–10.0 μg mL−1. Subsequently, by investigating the ability of different DNA sequences to restore the RLS intensity of the analytical systems, we found that ADM was selective to dsDNA and had an obvious preference for sequences that were rich in guanine and cytosine bases. In order to validate the results of the RLS assay, fluorescence quenching was used, and binding constants and binding numbers of each system were calculated. Compared with other methods, this RLS off–on strategy was more sensitive, fast, and reliable. It has also supplied a novel method for studying the sequence selectivity of DNA-targeted anticancer drugs and is a novel application of the RLS technique in analytical chemistry. Figure A resonance light scattering off-on system for the studies of selective interaction between adriamycin and DNA
Keywords: Anticancer drug; Resonance light scattering; Ag nanoparticle; DNA sequence; Selectivity

Rapid screening and quantification of sulfonate derivatives in white peony root by UHPLC–MS–MS by Zhixiang Yan; Chen Chen; Xiabing Xie; Bo Fu; Xinghao Yang (2173-2182).
A rapid ultra-high-performance liquid chromatographic–tandem mass spectrometric (UHPLC–MS–MS) method has been developed for rapid screening and quantitative analysis of sulfonate derivatives (SDs) in commercial white peony root. Separation was performed on an Agilent Zorbax Eclipse Plus-C18 column by gradient elution with acetonitrile–0.1% (v/v) formic acid as the mobile phase. In-source fragmentation was used to generate the characteristic fragment ion at m/z 259 and to screen for nine SDs. Detection of these SDs was further performed in multiple reaction monitoring (MRM) mode to improve sensitivity and to quantify the two SDs paeoniflorin sulfonate and benzoylpaeoniflorin sulfonate. The method was validated for specificity, linearity, limits of detection and quantification, precision, accuracy, and matrix effects. Nine commercial white peony root samples were examined by use of this method, which revealed great variety in the paeoniflorin sulfonate and benzoylpaeoniflorin sulfonate content. Figure Rapid screening of sulfonate derivatives in white peony root by extracted ion chromatogram (EIC) of m/z 259
Keywords: White peony root; Sulfonate derivatives; Ultra-high-performance liquid chromatography; Tandem mass spectrometry

Physico-chemical characterization of protein–pigment interactions in tempera paint reconstructions: casein/cinnabar and albumin/cinnabar by Celia Duce; Lisa Ghezzi; Massimo Onor; Ilaria Bonaduce; Maria Perla Colombini; Maria Rosaria Tine’; Emilia Bramanti (2183-2193).
In this work, we characterized paint reconstructions using ovalbumin and casein as binders, and cinnabar (HgS) as a pigment, before and after artificial ageing. Egg and casein are common paint binders that were used historically in the technique of tempera painting. Despite extensive research on the identification of proteinaceous binders in paintings, there is a substantial lack of knowledge regarding the ageing pathway of their protein content, and their chemical interaction with inorganic pigments. Thermogravimetric analysis, infrared spectroscopy and size-exclusion chromatography (SEC) were used to reveal the physico-chemical processes involved in the ageing of proteins in paintings. Taken together, the three techniques highlighted that proteins are subject to both cross-linking and hydrolysis upon ageing, and to a lesser extent, to oxidation of the side chains. Mercury–protein interactions were also revealed using a cold vapour generation atomic fluorescence spectrometer mercury-specific detector coupled to SEC. The study clearly showed that HgS forms stable complexes with proteins and acts as a sensitizer in cross-linking, hydrolysis and oxidation. Figure A multi-techinque approach to the study of protein/cinnabar tempera paint recontructions: thermogravimetric analysis, Fourier Transform Infrared Spectroscopy and size exclusion chromatography
Keywords: Ovalbumin; Casein; Cinnabar; Protein–pigment interaction; Cultural heritage; Size-exclusion chromatography; Fourier-transform infrared spectroscopy; Thermogravimetric analysis

In this paper, we propose an analytical methodology for attributing provenance to natural lapis lazuli pigments employed in works of art, and for distinguishing whether they are of natural or synthetic origin. A multitechnique characterization of lazurite and accessory phases in lapis lazuli stones from Afghan, Siberian and Chilean quarries, on the pigments obtained by their purification, and on synthetic ultramarine pigments was performed. According to the results obtained, infrared spectroscopy is not a suitable technique for distinguishing the provenance of lapis lazuli, but a particular absorbance band makes it relatively easy to determine whether it is of natural or synthetic origin. On the other hand, EDS elemental composition and XRD patterns show the presence of specific mineral phases associated with specific lapis lazuli sources, and can be used to distinguish the provenance of the stones as well as—albeit to a lesser extent—the corresponding purified blue pigments. In contrast, FEG-SEM observations clearly show different stone textures depending on their provenance, although these distinctive features do not persist in the corresponding pigments. PCA analyses of EDS data allow Afghan lapis lazuli stone to be distinguished from Chilean and Siberian ones, and can distinguish between the pigments resulting from their purification as well as synthetic blue ones. Although this methodology was developed using a limited number of samples, it was tested on lapis lazuli pigments collected from three paintings (from the fourteenth to sixteenth centuries) in order to perform a preliminary validation of the technique, and based on the results, the provenance of the blue pigments employed in those artworks is proposed. Finally, upon analytically monitoring the process of purifying lapis lazuli to obtain the corresponding pigments, it was found that ion-exchange reactions occur between the alkali modifiers of silicate/aluminosilicate phases and free carboxylic acids present in the doughy mixture of natural terpenes and ground stone, namely pastello. These reactions favor (i) the retention of silicate phases in the organic mixture and (ii) the selective extraction of lazurite due to the formation of Brønsted acidic sites [Al(OH)Si], which are responsible for its high hydrophilicity in comparison to the one of the other species present in the lapis lazuli stone. Figure Back scattered electron image of a polished section of Chilean lapis lazuli rock
Keywords: Lapis lazuli; Provenance; Pigment purification; FEG-ESEM-EDS; Paintings

A simple sample pretreatment technique, dispersive micro-solid phase extraction, was applied for the extraction of N-nitrosodimethylamine (NDMA) and other four N-nitrosamines (NAs) from samples of swimming pool water. The parameters affecting the extraction efficiency were systematically investigated. The best extraction conditions involved immersing 75 mg of carbon molecular sieve, Carboxen™ 1003 (as an adsorbent), in a 50-mL water sample (pH 7.0) containing 5% sodium chloride in a sample tube. After 20 min of extraction by vigorous shaking, the adsorbent was collected on a filter and the NAs desorbed by treatment with 150 μL of dichloromethane. A 10-μL aliquot was then directly determined by large-volume injection gas chromatography with chemical ionization mass spectrometry using the selected ion storage mode. The limits of quantitation were <0.9 ng/L. The precision for these analytes, as indicated by relative standard deviations, were <8% for both intra- and inter-day analyses. Accuracy, expressed as the mean extraction recovery, was between 62% and 109%. A preliminary analysis of swimming pool water samples revealed that NDMA was present in the highest concentration, in the range from n.d. to 100 ng/L.
Keywords: Water analysis; Dispersive micro-solid phase extraction; N-nitrosamines; GC-MS

Highly sensitive detection of microRNA by chemiluminescence based on enzymatic polymerization by Changbei Ma; Edward S. Yeung; Shengda Qi; Rui Han (2217-2220).
We have developed a new methodology for miRNA assay using chemiluminescence imaging by poly(U) polymerase catalyzed miRNA polymerization. This method is very sensitive with a 50 fM limit of detection, which is comparable to or better than current assay methods. Multiplex detection for miRNA can be easily realized by introducing different capture probes onto the biosensor array, which will make it highly versatile for various research purposes.
Keywords: MicroRNA; Chemiluminescence; Array

Nylon membrane as a fluorimetric probe for the herbicide bentazone by Jessica P. Chiarandini; Graciela M. Escandar (2221-2225).
The fluorimetric signal produced by bentazone retained in selected solid surfaces was investigated. Among the different tested supports, only a microporous nylon membrane produced the desired signal. The quantitative study was carried out by second-order calibration using parallel factor analysis, allowing the determination in a highly interfering medium. A detection limit of 0.4 ng mL−1, a prediction relative error of 8%, and a sample frequency of ten samples per hour were obtained in spiked natural waters using green analytical chemistry principles.
Keywords: Optical fluorimetric probe; Nylon membrane; Second-order calibration; Bentazone

Erratum to: Analytical method for assessing potential dermal exposure to pesticides of a non-agricultural occupationally exposed population by Olivier Delhomme; Caroline Raeppel; Delphine Teigné; Olivier Briand; Maurice Millet (2227-2228).