Analytical and Bioanalytical Chemistry (v.392, #4)

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 retraining as a university careers adviser, in 2000, she founded a company ( http://www.shintonconsulting.com ) and now delivers careers guidance and skills training to academic researchers and scientists across the UK and Europe.

is Centennial Professor of Chemistry, Emeritus at Vanderbilt University. His current research is on mass spectrometry of synthetic polymers, primarily using MALDI as the technique of choice. Special emphasis is on the use of MS-MS for analytical applications and to study polymer decomposition mechanisms. A second major emphasis is on MALDI of insoluble polymers. He is also investigating the use of IMS/MALDI for polymer analysis.

The detailed structural characterization of complex polymer architectures, like copolymers and polymer mixtures, by mass spectrometry presents a challenge. Even though soft ionization analyses revolutionized the characterization of large molecules and provided a means for determining the polymer’s molecular weight distribution, polydispersity, and end groups, full microstructure elucidation and monomer sequencing by soft ionization alone is not possible. The combination of high-resolution Fourier transform mass spectrometry (FTMS) and tandem mass spectrometry (MSn) provides a powerful analytical tool for addressing these challenges. This tool was used in our work to separate and identify the products of polymerization between 12-hydroxystearic acid (HSA) and stearic acid (SA), to provide precise information about the exact location of caprolactones on the Tris(2-hydroxyethyl)isocyanurate (THEIC) molecule, and to sequence a glycidyl methacrylate/methyl methacrylate (GMA/MMA) copolymer. The results highlight the value of ultrahigh resolution and tandem mass spectrometry for fine structural characterization and sequencing of polymers.
Keywords: Electrospray ionization; Laser desorption ionization; Fourier transform mass spectrometry; Collision-induced dissociation; Polymers; Copolymers; Tandem mass spectrometry

A wide mass range trapping experiment using internal source matrix-assisted laser desorption–Fourier transform mass spectrometry (MALDI–FTMS) was evaluated. In this method, the front trap plate potential is ramped up and the rear trap plate potential is simultaneously decreased using a cubic cell to trap ions over a wide range of mass-to-charge ratios. To apply this to MS/MS experiments, a second ion ejection procedure would remove unwanted ions, with the selected remaining ions then fragmented by collision-induced dissociation. In measurements using a 7.2-T unshielded magnet presented here, an approximately equimolar mixture of a set of poly(ethylene glycol) (PEG) species for the ramped measurements had peak areas of 1.0:1.0:1.0:1.0, as did the previously described integral method which gave peak areas of 1.0:1.1:1.0:1.0, in good agreement with the known composition of the samples deposited on the MALDI probe tip. Comparative MALDI–TOF in reflectron mode results were of similar quality for the equimolar mixture, giving a ratio of 1.0:1.0:1.2:0.9. All methods failed to varying degrees when individual PEG compositions of the trial mixture were changed. However, the previously described integral method showed relatively better results for all but the PEG 8000 doubled mixture.
Keywords: SORI–FTMS dissociation; Matrix-assisted laser desorption; Fourier transform mass spectrometry; Wide mass range polymer analysis

Characterization of linear and branched polyacrylates by tandem mass spectrometry by Kittisak Chaicharoen; Michael J. Polce; Anirudha Singh; Coleen Pugh; Chrys Wesdemiotis (595-607).
The unimolecular degradation of alkali-metal cationized polyacrylates with the repeat unit CH2CH(COOR) and a variety of ester pendants has been examined by tandem mass spectrometry. The fragmentation patterns resulting from collisionally activated dissociation depend sensitively on the size of the ester alkyl substituent (R). With small alkyl groups, as in poly(methyl acrylate), lithiated or sodiated oligomers (M) decompose via free-radical chemistry, initiated by random homolytic C-C bond cleavages along the polymer chain. The radical ions formed this way dissociate further by backbiting rearrangements and β scissions to yield a distribution of terminal fragments with one of the original end groups and internal fragments with 2–3 repeat units. If the ester alkyl group bears three or more carbon atoms, cleavages within the ester moieties become the predominant decomposition channel. This distinct reactivity is observed if R = t-butyl, n-butyl, or the mesogenic group (CH2)11-O-C6H4-C6H4-CN. The [M+alkali metal]+ ions of the latter polyacrylates dissociate largely by charge-remote 1,5-H rearrangements that convert COOR to COOH groups by expulsion of 1-alkenes. The acid groups may displace an alcohol unit from a neighboring ester pendant to form a cyclic anhydride, unless hindered by steric effects. Using atom transfer radical polymerization, hyperbranched polyacrylates were prepared carrying ester groups both within and between the branches. Unique alkenes and alcohols are cleaved from ester groups at the branching points, enabling determination of the branching architecture. Figure MALDI-CAD tandem mass spectrum of the lithiated 4-mer from a hyperbranched polyacrylate. The fragments marked by green stars diagnose the branched architecture shown on top of the spectrum. The fragments marked by violet stars diagnose a different isomer.
Keywords: Polyacrylate fragmentation; Degradation mechanisms; Tandem mass spectrometry; Mesogenic substituents; Hyperbranched polyacrylates; Branching architecture; Charge-remote rearrangement

MALDI-TOF/TOF CID study of 4-alkyl-substituted polystyrene fragmentation reactions by Anthony P. Gies; Matthew J. Vergne; Rebecca L. Orndorff; David M. Hercules (609-626).
MALDI-TOF/TOF CID experiments were conducted on a variety of hydrogen-terminated poly(4-methylstyrene), hydroxylated poly(t-butylstyrene), and polystyrene precursor ions: n = 10, 15, 20, 25, and 30, where the number of repeat units n corresponds to the oligomer mass number. The influences of structure, molecular weight, and effective collision kinetic energy on degradation mechanisms were examined to test the generality of our multi-chain fragmentation model developed for polystyrene. Each depolymerization mechanism is presented in detail with experimental and computational data to justify/rationalize its occurrence and effective kinetic energy dependence. These processes show the complex interrelationship between the various pathways along with preferred production of secondary radicals, which suppresses the appearance of primary radicals. Additionally, Py-GC/MS experimental data are presented, for comparison of the multimolecular free radical reactions in pyrolysis with the unimolecular fragmentation reactions of MS/MS.
Keywords: MALDI-TOF/TOF CID mass spectrometry; Polymers; Modeling; MS/MS; Poly(t-butylstyrene); Poly(4-methylstyrene)

MALDI-TOF/TOF CID study of poly(α-methylstyrene) fragmentation reactions by Anthony P. Gies; Sparkle T. Ellison; Matthew J. Vergne; Rebecca L. Orndorff; David M. Hercules (627-642).
MALDI-TOF/TOF CID experiments are reported for hydroxylated poly(α-methylstyrene) precursor ions (PAMS: m/z 1,445.9 (n = 10), 2,036.3 (n = 15), 2,626.7 (n = 20), 3,217.1 (n = 25), and 3,807.5 (n = 30), where the number of repeat units n corresponds to the oligomer mass numbers). The influences of structure, molecular weight, and kinetic energy on degradation mechanisms were examined to test the generality of our multi-chain fragmentation model developed for polystyrene. Our results indicate that poly(α-methylstyrene) free radicals are formed initially through multiple chain breaks and subsequently undergo a variety of depolymerization reactions to yield predominantly monomer and dimer species; the intensity of each species depends on the effective kinetic energy selected for the CID process. Each depolymerization mechanism is presented in detail with experimental and computational data to justify/rationalize the process and its kinetic energy dependence. These processes show the complex interrelationships between the various pathways along with preferred production of tertiary radicals, which suppresses the appearance of primary radicals. Additionally, Py-GC/MS experimental data are presented to allow a comparison of the multimolecular free radical reactions in pyrolysis with the unimolecular fragmentation reactions of MS/MS.
Keywords: MALDI-TOF/TOF CID; Modeling; Styrenic polymers; Mass spectrometry; Fragmentation mechanisms; Pyrolysis

End-group characterisation of poly(propylene glycol)s by means of electrospray ionisation–tandem mass spectrometry (ESI-MS/MS) by Anthony T. Jackson; Susan E. Slade; Konstantinos Thalassinos; James H. Scrivens (643-650).
The end-group functionalisation of a series of poly(propylene glycol)s has been characterised by means of electrospray ionisation–tandem mass spectrometry (ESI-MS/MS). A series of peaks with mass-to-charge ratios that are close to that of the precursor ion were used to generate information on the end-group functionalities of the poly(propylene glycol)s. Fragment ions resulting from losses of both of the end groups were noted from some of the samples. An example is presented of how software can be used to significantly reduce the length of time involved in data interpretation (which is typically the most time-consuming part of the analysis). Figure Screenshot from Polymerator software of annotated ESI-MS/MS spectrum from the lithiated heptamer of poly(propylene glycol) di-acrylate
Keywords: Polyether; PPG; End groups; Electrospray ionisation; Tandem mass spectrometry; Software

Electrochemical detection of acetylcholine and choline: application to the quantitative nonradiochemical evaluation of choline transport by Tatyana V. Barkhimer; Jon R. Kirchhoff; Richard A. Hudson; William S. Messer Jr; L. M. Viranga Tillekeratne (651-662).
The development of analytical methods for determining the cholinergic biomarkers acetylcholine (ACh) and choline (Ch) is important for assessing their role in neurological and cognitive functions. In this review, electrochemical (EC) strategies to detect ACh and Ch are summarized and compared to other analysis methods. Recent research focusing on the development of a versatile nonradiochemical in vitro assay to evaluate Ch transport is also described. The assay coupled to analysis by capillary electrophoresis (CE) with indirect EC detection at an enzyme-modified microelectrode affords exceptional selectivity and sensitivity. Femtomole or lower mass detection limits for ACh (1 fmol) and Ch (100 amol) have been readily achieved, opening up a new range of possible experiments for investigating transport or turnover of Ch and ACh in neurobiological systems. The value of this method is illustrated through the evaluation of the pharmacological efficacy and mode of inhibition of a new class of quaternary ammonium alkyl-substituted catechol-based inhibitors of high-affinity choline transport (CHT). This microanalytical approach is particularly useful when knowledge of endogenous concentrations of Ch or ACh is desired or when the amount of available compounds or the sample size is limited. A brief description of the principles of CE is also provided.
Keywords: Acetylcholine; Choline; Capillary electrophoresis with electrochemical detection; Enzyme microelectrodes; High-affinity choline transport; Cholinergic inhibition

Comparison of high-resolution and dynamic reaction cell ICP-MS capabilities for forensic analysis of iron in glass by Waleska Castro; Tatiana Trejos; Benjamin Naes; José R. Almirall (663-672).
Forensic laboratories routinely conduct analysis of glass fragments to determine whether or not there is an association between a fragment(s) recovered from a crime scene or from a suspect to a particular source of origin. The physical and optical (refractive index) properties of the fragments are compared and, if a “match” between two or more fragments is found, further elemental analysis can be performed to enhance the strength of the association. A range of spectroscopic techniques has been used for elemental analysis of this kind of evidence, including inductively coupled plasma mass spectrometry (ICP-MS). Because of its excellent sensitivity, precision, and accuracy, several studies have found that ICP-MS methods (dissolution and laser-ablation) provide the best discrimination between glass fragments originating from different sources. Nevertheless, standard unit-resolution ICP-MS instruments suffer from polyatomic interferences including 40Ar16O+, 40Ar16O1H+, and refractory oxide 40Ca16O+ that compromise measurements of trace levels of Fe56+ and Fe57+, for example. This is a drawback in the analysis of glass fragments because iron has been previously identified as a good discriminating element. Currently, several techniques are available that enable reduction of such interferences. However, there are no data comparing detection limits of iron in glass using those techniques. The aim of this study was to compare, the analytical performance of high-resolution sector field inductively coupled plasma mass spectrometry (HR-SF-ICP-MS) and quadrupole ICP-MS equipped with a dynamic reaction cell (DRC-ICP-MS), for the detection of iron in glass, in terms of accuracy, precision, and method detection limits (MDLs). Analyses were conducted using conventional acid-digestion and laser-ablation methods. For laser-ablation analyses, carrier gases were compared to assess the effect on detection limits in the detection of iron isotopes. Iron polyatomic interferences were reduced or resolved by using a dynamic reaction cell and high-resolution ICP-MS. MDLs as low as 0.03 μg g−1 and 0.14 μg g−1 were achieved in laser-ablation and solution-based analyses, respectively. Use of helium as carrier gas improved detection limits for both iron isotopes in medium-resolution HR-SF-ICP-MS and in DRC-ICP-MS.
Keywords: Glass; Elemental analysis; DRC-ICP-MS; HR-SF-ICP-MS; Forensic

Farnesyl- and geranylgeranylpyrophosphate (FPP and GGPP) are isoprenoid intermediates in the mevalonate pathway. They play a crucial role in cell survival, growth and differentiation due to their attachment (isoprenylation) to small GTPases (Ras, Rho, etc.). Isoprenoid formation seems to be tightly regulated within the mevalonate pathway and its perturbation has been linked to certain diseases (e.g., cancer, Alzheimer’s disease), but tissue levels are unknown. It is therefore of the utmost importance to quantify these isoprenoids in diseased tissue or in tissue after drug administration. The current work describes an isolation procedure utilizing a combination of Extrelut® liquid/liquid and reversed-phase solid-phase extraction (SPE) for homogenized human frontal cortex tissue. In addition, after a careful validation of an HPLC–fluorescence method, this assay allowed the determination of nanomolar concentrations of endogenous FPP and GGPP levels (4.5 and 10.6 ng/mg protein, respectively) in human brain tissue. The method is selective, precise (<15% RSD), accurate (<15% relative error) and sensitive over a linear range of 10–400 ng/mL for FPP and 50–1000 ng/mL for GGPP according to the current FDA criteria for bioanalytical method validation. Overall, this new method introduces the ability to simultaneously quantify FPP and GGPP in human brain tissue, and is potentially applicable to several other tissues and species.
Keywords: Isoprenylation; Farnesylpyrophosphate (FPP); Geranylgeranylpyrophosphate (GGPP); Alzheimer’s disease (AD); Cancer; High-performance liquid chromatography (HPLC); Validation

This paper presents a general screening method, based on liquid chromatography/mass spectrometry (LC/MS), for the simultaneous detection in human urine of 72 xenobiotics (21 diuretics, 16 synthetic glucocorticoids, 17 beta-adrenergic drugs, 10 stimulants, 5 anti-oestrogens and 3 anabolic steroids), excreted free or as glucuro-conjugates in urine. Although the method has been specifically designed and evaluated in view of its potential application to anti-doping analyses, it can also be effective in other areas of analytical toxicology. Sample preparation was based on two liquid/liquid separation steps (performed at alkaline and at acid pH, respectively) of hydrolyzed human urine, and then an assay by LC/MS-MS in positive and negative ionization mode using an electrospray ionization source (ESI) and multiple reaction monitoring (MRM) as the acquisition mode. The overall time needed for an LC run was less than 15 minutes. All compounds showed good reproducibility in terms of both the retention times (CV%<1) and the relative abundances of the diagnostic transitions (CV%<10). The limits of detection (LOD) were in the range of 1–50 ng/mL for glucocorticoids, anti-oestrogens and steroids, and 50–500 ng/mL for diuretics, beta-adrenergic drugs and stimulants, thus satisfying the minimum required performance limits (MRPL) set by the World Anti-Doping Agency (WADA) for the accredited anti-doping laboratories.
Keywords: Glucocorticoids; Anti-oestrogens; Designer steroids; Stimulants; Diuretics; Beta-adrenergic drugs; LC-ESI-MS/MS; Anti-doping analysis

Acetylcholinesterase-based biosensors for quantification of carbofuran, carbaryl, methylparaoxon, and dichlorvos in 5% acetonitrile by Gabriela Valdés-Ramírez; Montserrat Cortina; Maria Teresa Ramírez-Silva; Jean-Louis Marty (699-707).
Amperometric acetylcholinesterase biosensors have been developed for quantification of the pesticides carbofuran, carbaryl, methylparaoxon, and dichlorvos in phosphate buffer containing 5% acetonitrile. Three different biosensors were built using three different acetylcholinesterase (AChE) enzymes—AChE from electric eel, and genetically engineered (B394) and wild-type (B1) AChE from Drosophila melanogaster. Enzymes were immobilized on cobalt(II) phthalocyanine-modified electrodes by entrapment in a photocrosslinkable polymer (PVA-AWP). Each biosensor was tested against the four pesticides. Good operational stability, immobilisation reproducibility, and storage stability were obtained for each biosensor. The best detection limits were obtained with the B394 enzyme for dichlorvos and methylparaoxon (9.6 × 10−11 and 2.7 × 10−9 mol L−1, respectively), the B1 enzyme for carbofuran (4.5 × 10−9 mol L−1), and both the B1 enzyme and the AChE from electric eel for carbaryl (1.6 × 10−7 mol L−1). Finally, the biosensors were used for the direct detection of the pesticides in spiked apple samples.
Keywords: Acetylcholinesterase; Carbofuran; Carbaryl; Methylparaoxon; Dichlorvos; Acetonitrile–PBS

Site-specific binding of chelerythrine and sanguinarine to single pyrimidine bulges in hairpin DNA by Li-Ping Bai; Zongwei Cai; Zhong-Zhen Zhao; Kazuhiko Nakatani; Zhi-Hong Jiang (709-716).
Spectrofluorometric titration, electrospray ionization time-of-flight mass spectrometric and UV melting methods were employed to study the binding of chelerythrine and sanguinarine to bulged DNA. The results showed that both alkaloids bind specifically to single pyrimidine (C, T) bulge sites. The ability of sanguinarine to bind to both regular and bulged hairpins was found to be stronger than that of chelerythrine, but the binding selectivity of chelerythrine toward single-base bulges was much larger than that of sanguinarine. Figure Association constants for chelerythrine and sanguinarine toward regular and single-base bulged hairpins obtained from fluorometric analysis
Keywords: Chelerythrine; Sanguinarine; Bulge DNA; Fluorescence spectrometry; ESI-MS

Despite first evidence for the cytochrome P450-mediated enantioselective biosynthesis and activity of cis-epoxyeicosatrienoic acids (EETs), as yet little is known about the stereospecifity of EET generation and physiology, because the existing chiral methods are time consuming, labor intensive, and not sensitive enough. We present a method for highly sensitive, direct, and simultaneous chiral analysis of all eight EET enantiomers consisting of (i) solid-phase extraction, (ii) reversed-phase high-performance liquid chromatographic purification followed by (iii) consecutive regio- and enantiomeric separation of the four underivatized EET regioisomers within one chromatographic run employing capillary tandem column chiral-phase liquid chromatography with (iv) reliable dual online photodiode array and gentle electrospray ionization tandem mass spectrometric identification and quantitation of the eluting optical antipodes. This one-step, simple, expeditious, and highly sensitive measurement allows profiling of all eight EET enantiomers at once, thus avoiding substance loss and enabling high sample throughput. Limits of quantification in the low picogram range were achieved by the use of capillary columns with typical high quantitative sensitivity instead of conventional columns with low chromatographic signal intensity employed by previous methods. Application to tissue homogenates demonstrated the suitability of this approach for routine and reliable “enantioprofiling” of free endogenous EETs, i.e., EETs not esterified into cellular membrane phospholipids, typically occurring at very low concentrations. The technique can readily be employed for preparative purification of enantiomers in the microgram range using large-inner-diameter columns. Figure Direct and simultaneous enantioprofiling of the four free endogenous epoxyeicosatrienoic acids (EETs) from a complex biological matrix, like the cardiopulmonary system, within one chromatographic run by highly sensitive, one-step capillary tandem column chiral-phase liquid chromatography with dual online photodiode array and tandem mass spectrometric detection (CapTC-CP-LC-PDAD-ESI-MS2) enables accurate, systematic, and routine correlation between the absolute configuration of EETs and their physiological actions
Keywords: Profiling of epoxyeicosatrienoic acid enantiomers; Capillary tandem column chiral-phase liquid chromatography; Photodiode array detection; Electrospray ionization tandem mass spectrometry; Multiple reaction monitoring; Extracted ion chromatogram

Development and characterization of new rat monoclonal antibodies for procalcitonin by Petra M. Krämer; Marie-Françoise Gouzy; Melanie Keß; Ulrike Kleinschmidt; Elisabeth Kremmer (727-736).
The development of selective and sensitive biological recognition elements, e.g., antibodies, for the detection of relevant blood markers is a great challenge in the field of biosensors. In this context, five new rat monoclonal antibodies (mAbs) for procalcitonin (PCT), a marker for bacterial infection and sepsis, were developed and characterized. One mAb, PROC1 3G3, was used as capture antibody. Four mAbs, PROC4 6C6, PROC4 6B2, PROC4 1G3, and PROC4 1D6, were used as detection mAbs, either as Protein G-purified or as biotinylated mAbs. A surface plasmon resonance (SPR) biosensor was used to characterize the antigen–antibody biomolecular interactions. The capture mAb (PROC1 3G3) has an equilibrium dissociation constant (K D) of 3.42 × 10–8 M. All four detection mAbs (PROC4 6C6, PROC4 6B2, PROC4 1G3, and PROC4 1D6) are of high affinity (K A = 2.81–6.11 × 108 M−1; K D = 1.64–3.56  × 10–9 M) and have moderate dissociation rate constants (k d = 1.70–2.40 × 10–3 s−1). Four different sandwich enzyme-linked immunosorbent assays (ELISAs) with standards of human recombinant (hr) PCT, using PROC1 3G3 as capture mAb and PROC4 mAbs as detection mAbs, respectively, led to highly specific determinations of PCT without cross-reactivities to calcitonin and katacalcin. The lower limits of quantification (LLOQ) for hrPCT (in 40 mM phosphate-buffered saline (PBS), pH 7.6) with these assays ranged from 2.3 to 12.8 µg L−1. In addition, sandwich ELISAs were set up with biotinylated PROC4 mAbs, and with hrPCT in 4% human serum albumin (diluted 1:10 in 40 mM PBS, including 1:5 (v/v) LowCross Buffer®). The LLOQs of these sandwich assays ranged from 4.1 to 6.0 µg L−1 and were thus much closer together for the different assays. With the latter assay setup (PROC1 3G3 as capture mAb, PROC4 6C6–biotin as detection mAb) a first collection of five serum samples was determined (healthy volunteers, unspiked, and spiked). Recovery rates for the spiked samples ranged from 98.3 to 115.7%. The newly developed anti-PCT mAbs should find broad applications in immunosensors for point-of-care diagnostics of sepsis and systemic inflammation processes.
Keywords: Procalcitonin; Katacalcin; Calcitonin; Monoclonal antibody; Sandwich ELISA; Surface plasmon resonance

A multi-isotope spike species-specific isotope dilution (MI-SSID) calibration strategy in connection with gas chromatography-inductively coupled plasma mass spectrometry was applied to evaluate different extraction procedures for the speciation analysis of phenyltin (PhT) compounds in biological materials: mussel tissue BCR CRM-477 and fish tissue NIES-11. Three different isotope-enriched PhT compounds, 118Sn-enriched monophenyltin (MPhT), 122Sn-enriched diphenyltin (DPhT), and 124Sn-enriched triphenyltin (TPhT), were used for the preparation of spikes to follow and correct for six possible interconversion reactions between PhT species that can take place in a sample. The acidity of the extractant, the presence of complexing reagents, and the use of ultrasonic or microwave agitation were found to affect the degradation of PhT compounds. No formation of PhTs through phenylation and negligible degradation of MPhT to inorganic tin were observed under the conditions investigated. The degree of degradation increased with increased acidity of extractant and when ultrasonication or microwave agitation was used. Under relatively mild extraction conditions, the degradation factors for DPhT and TPhT in the two reference materials studied were found, using MI-SSID, to be between 10 and 55% and 2 and 10%, respectively. Using the degradation factors, we calculated corrected concentration values for the organotin species. When microwave extraction at high power output was used, hydrogen radicals were formed that can enhance the degradation of DPhT and TPhT. The hydrogen radicals were trapped using N-tert-butyl-α-phenylnitrone and detected by electron spin resonance spectrometry. The effect of different extraction parameters on the degradation of PhT compounds in biological samples is discussed.
Keywords: Biological samples; Microwave digestion; Gas chromatography; Inductively coupled plasma mass spectrometry; Speciation; Extraction

Analysis of captan, folpet, and captafol in apples by dispersive liquid–liquid microextraction combined with gas chromatography by Xiaohuan Zang; Juntao Wang; Ou Wang; Mingzhao Wang; Jingjun Ma; Guohong Xi; Zhi Wang (749-754).
A novel method was developed for the determination of captan, folpet, and captafol in apples by dispersive liquid–liquid microextraction (DLLME) coupled with gas chromatography–electron capture detection (GC–ECD). Some experimental parameters that influence the extraction efficiency, such as the type and volume of the disperser solvents and extraction solvents, extraction time, and addition of salt, were studied and optimized to obtain the best extraction results. Under the optimum conditions, high enrichment factors for the compounds were achieved ranging from 824 to 912. The recoveries of fungicides in apples at spiking levels of 20.0 μg kg−1 and 70.0 μg kg−1 were 93.0–109.5% and 95.4–107.7%, respectively. The relative standard deviations (RSDs) for the apple samples at 30.0 μg kg−1 of each fungicide were in the range from 3.8 to 4.9%. The limits of detection were between 3.0 and 8.0 μg kg−1. The linearity of the method ranged from 10 to 100 μg kg−1 for the three fungicides, with correlation coefficients (r 2) varying from 0.9982 to 0.9997. The obtained results show that the DLLME combined with GC–ECD can satisfy the requirements for the determination of fungicides in apple samples. Figure Dispersive liquid–liquid microextraction (DLLME) coupled with gas chromatography–electron capture detection (GC–ECD) allows satisfactory determination of fungicides in apple samples
Keywords: Captan; Folpet; Captafol; Apple; GC–ECD; Dispersive liquid–liquid microextraction

Multianalytical approach to the analysis of English polychromed alabaster sculptures: μRaman, μEDXRF, and FTIR spectroscopies by Kepa Castro; Alfredo Sarmiento; Maite Maguregui; Irantzu Martínez-Arkarazo; Nestor Etxebarria; Manuel Angulo; María Urrutikoetxea Barrutia; Juan Manuel González-Cembellín; Juan Manuel Madariaga (755-763).
A complete study of several English polychromed alabaster sculptures is presented. The support, pigment, and binders were characterised by combining μEDXRF, μRaman, and FTIR spectroscopies. Among the pigments, minium, vermilion, lead white, carbon black, red iron oxide, and a degraded green copper pigment were determined, together with gold leaf. The presence of the rare mineral moolooite (copper oxalate) was also found as a degradation product in the green areas, where weddellite (calcium oxalate dihydrate) was also determined. These facts, together with degradation of the green copper pigment, suggest microbiological degradation of the original materials. Remains of glue and a varnish were also determined by FTIR spectroscopy and principal-components analysis (PCA) of the spectra. Finally, PCA analysis was carried out to confirm whether the pieces came from the same quarry.
Keywords: Pigment; Alabaster; Raman; XRF; Artwork; Sculpture