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Analytical and Bioanalytical Chemistry (v.403, #5)

Industrial research and development for instrumental analytics: requirements, skills, strategic objectives by Gerhard Schlemmer (pp. 1195-1198).

has been working with suppliers of analytical equipment for almost 30 years. During this time he has been in various functions, from applied research to product management, from product and customer support to market development. During the past 10 years he was head of the R&D department of a German manufacturer of analytical instruments. Since end of 2011 he is consulting in the field of project management, technology transfer and application for instrumental analytics.

Teresa Cecchi: Ion-pair chromatography and related techniques by Bogusław Buszewski (pp. 1199-1200).

High-resolution mass spectrometry by Hans H. Maurer; David C. Muddiman (pp. 1201-1202).

is the Head of the Department of Experimental and Clinical Toxicology of Saarland University in Homburg. He has published over 200 original papers and over 20 invited reviews on his main two areas of research, analytical toxicology (GC–MS, LC–MS) and metabolism of xenobiotics. He received several international scientific awards for his outstanding scientific work, and in 2007 he was awarded the title of Doctor Honoris Causa (honorary doctorate) by the University of Ghent, Belgium. is currently a Professor of Chemistry and Founder and Director of the W.M. Keck FT-ICR Mass Spectrometry Laboratory at North Carolina State University in Raleigh, NC, USA. His group has presented over 350 invited lectures and presentations at national and international meetings, has published over 195 peer-reviewed papers, and has been awarded three US patents. He is the recipient of the 2010 Biemann Medal of the American Society for Mass Spectrometry, the 2009 NCSU Alumni Outstanding Research Award, the 2004 ACS Arthur F. Findeis Award, the 1999 American Society for Mass Spectrometry Research Award, and the Safford Award, University of Pittsburgh, for Excellence in Teaching.

Current use of high-resolution mass spectrometry in drug screening relevant to clinical and forensic toxicology and doping control by Ilkka Ojanperä; Marjo Kolmonen; Anna Pelander (pp. 1203-1220).

Clinical and forensic toxicology and doping control deal with hundreds or thousands of drugs that may cause poisoning or are abused, are illicit, or are prohibited in sports. Rapid and reliable screening for all these compounds of different chemical and pharmaceutical nature, preferably in a single analytical method, is a substantial effort for analytical toxicologists. Combined chromatography–mass spectrometry techniques with standardised reference libraries have been most commonly used for the purpose. In the last ten years, the focus has shifted from gas chromatography–mass spectrometry to liquid chromatography–mass spectrometry, because of progress in instrument technology and partly because of the polarity and low volatility of many new relevant substances. High-resolution mass spectrometry (HRMS), which enables accurate mass measurement at high resolving power, has recently evolved to the stage that is rapidly causing a shift from unit-resolution, quadrupole-dominated instrumentation. The main HRMS techniques today are time-of-flight mass spectrometry and Orbitrap Fourier-transform mass spectrometry. Both techniques enable a range of different drug-screening strategies that essentially rely on measuring a compound’s or a fragment’s mass with sufficiently high accuracy that its elemental composition can be determined directly. Accurate mass and isotopic pattern acts as a filter for confirming the identity of a compound or even identification of an unknown. High mass resolution is essential for improving confidence in accurate mass results in the analysis of complex biological samples. This review discusses recent applications of HRMS in analytical toxicology.

Keywords: High-resolution mass spectrometry; Drug screening; Time-of-flight; Orbitrap; Toxicology; Doping

Current applications of high-resolution mass spectrometry in drug metabolism studies by Markus R. Meyer; Hans H. Maurer (pp. 1221-1231).

This paper reviews high-resolution mass spectrometry (HRMS) approaches published in 2007–2011 for the elucidation of drug metabolism with a focus on new therapeutics, new drugs of abuse, and doping agents using time-of-flight, single-stage Orbitrap, ion trap Orbitrap, and other Fourier transform MS-based techniques. The present review provides an overview of metabolite-generating systems and assays used, sample preparation techniques, ionization and fragmentation techniques, as well as data mining strategies and software tools which were used in the reviewed papers. Furthermore, HRMS-specific topics such as demand for a certain resolution or a specific mass accuracy are discussed in detail and corresponding recommendations are given. Finally, the advantages and limitations of these methods are discussed.

Keywords: High resolution; Mass spectrometry; Liquid chromatography; Gas chromatography; Metabolism

The current role of high-resolution mass spectrometry in food analysis by Anton Kaufmann (pp. 1233-1249).

High-resolution mass spectrometry (HRMS), which is used for residue analysis in food, has gained wider acceptance in the last few years. This development is due to the availability of more rugged, sensitive, and selective instrumentation. The benefits provided by HRMS over classical unit-mass-resolution tandem mass spectrometry are considerable. These benefits include the collection of full-scan spectra, which provides greater insight into the composition of a sample. Consequently, the analyst has the freedom to measure compounds without previous compound-specific tuning, the possibility of retrospective data analysis, and the capability of performing structural elucidations of unknown or suspected compounds. HRMS strongly competes with classical tandem mass spectrometry in the field of quantitative multiresidue methods (e.g., pesticides and veterinary drugs). It is one of the most promising tools when moving towards nontargeted approaches. Certain hardware and software issues still have to be addressed by the instrument manufacturers for it to dislodge tandem mass spectrometry from its position as the standard trace analysis tool.

Keywords: High-resolution mass spectrometry; Food analysis; Oribitrap; Time-of-flight mass spectrometry; Residue analysis

Current use of high-resolution mass spectrometry in the environmental sciences by F. Hernández; J. V. Sancho; M. Ibáñez; E. Abad; T. Portolés; L. Mattioli (pp. 1251-1264).

During the last two decades, mass spectrometry (MS) has been increasingly used in the environmental sciences with the objective of investigating the presence of organic pollutants. MS has been widely coupled with chromatographic techniques, both gas chromatography (GC) and liquid chromatography (LC), because of their complementary nature when facing a broad range of organic pollutants of different polarity and volatility. A clear trend has been observed, from the very popular GC–MS with a single quadrupole mass analyser, to tandem mass spectrometry (MS–MS) and, more recently, high-resolution mass spectrometry (HRMS). For years GC has been coupled to HR magnetic sector instruments, mostly for dioxin analysis, although in the last ten years there has been growing interest in HRMS with time-of-flight (TOF) and Orbitrap mass analyzers, especially in LC–MS analysis. The increasing interest in the use of HRMS in the environmental sciences is because of its suitability for both targeted and untargeted analysis, owing to its sensitivity in full-scan acquisition mode and high mass accuracy. With the same instrument one can perform a variety of tasks: pre- and post-target analysis, retrospective analysis, discovery of metabolite and transformation products, and non-target analysis. All these functions are relevant to the environmental sciences, in which the analyst encounters thousands of different organic contaminants. Thus, wide-scope screening of environmental samples is one of the main applications of HRMS. This paper is a critical review of current use of HRMS in the environmental sciences. Needless to say, it is not the intention of the authors to summarise all contributions of HRMS in this field, as in classic descriptive reviews, but to give an overview of the main characteristics of HRMS, its strong potential in environmental mass spectrometry and the trends observed over the last few years. Most of the literature has been acquired since 2005, coinciding with the growth and popularity of HRMS in this field, with a few exceptions that deserve to be mentioned because of their relevance.

Keywords: Environmental analysis; Organic contaminants; High-resolution mass spectrometry; Time-of-flight; Magnetic sector; Orbitrap; Liquid chromatography; Gas chromatography

Combined drug screening and confirmation by liquid chromatography time-of-flight mass spectrometry with reverse database search by Ana de Castro; Merja Gergov; Pekka Östman; Ilkka Ojanperä; Anna Pelander (pp. 1265-1278).

A method based on in-source collision-induced dissociation (ISCID) liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) and reverse target database search was developed and evaluated for drug screening and confirmation in analytical toxicology context. An established LC-TOFMS screening method, in which identification relies solely on protonated molecule accurate mass measurement, isotopic pattern fit, and retention time (RT), was completed to include 1–3 qualifier ions for each analyte in the database. The qualifier ions for 431 compounds were selected from the experimental ISCID spectra, and their molecular formulae were assigned by applying SmartFormula3D and MSFragmenter software. Three qualifier ions were assigned for 64.5%, two or three for 81.4%, one for 14.8%, and none for 3.7% of the compounds studied. Comparison between ISCID LC-TOFMS and LC-TOFMS with 25 authentic autopsy urine samples showed an improved confidence level with the ISCID method, as isomeric interferences were excluded in most cases. However, some false negative (FN) results were obtained at low concentration levels close to the reporting criteria. The cut-off concentration of the ISCID method was 10–100 ng/mL with 80% of the 49 representative compounds tested, and the level was approximately two times higher than that obtained by LC ion trap MS. The presented method enables simultaneous screening and confirmation whenever at least one qualifier ion is available, as applying an accurate mass precursor ion and one product ion surpasses the standard of four identification points that is required by the current EU protocol.

Keywords: LC-TOFMS; Drug screening; Confirmation; Identification

Sensitive determination of prohibited drugs in dried blood spots (DBS) for doping controls by means of a benchtop quadrupole/Orbitrap mass spectrometer by Andreas Thomas; Hans Geyer; Wilhelm Schänzer; Catharina Crone; Markus Kellmann; Thomas Moehring; Mario Thevis (pp. 1279-1289).

In the present study, a new type of mass spectrometer combining a quadrupole mass filter, a higher collision dissociation (HCD) cell and an Orbitrap detector, was evaluated for the analysis of dried blood spots (DBS) in doping controls. DBS analysis is characterized by the necessity to detect prohibited compounds in sub-nanogram-per-milliliter levels with high identification capacity. After extraction of DBS with an organic solvent and liquid chromatographic separation (using a regular C18-RP-analytical UHPLC-column) of target analytes, mass spectrometry is performed with a high-resolution full scan in positive and negative mode by means of electrospray ionisation. Single-product ion mass spectra are acquired using the data-dependent analysis mode (employing an inclusion list) for previously selected precursors of known prohibited compounds with fixed retention time ranges. Besides, a sensitive screening in a targeted approach, non-targeted analysis for retrospective data evaluation is thus possible. The chosen experimental design enables the determination of various drugs from different classes with one generic sample preparation which is shown for 26 selected model compounds (Δ⁹-tetrahydrocannabinol (THC), tetrahydrocannabinol-9-carboxylic acid (THC-COOH), methylhexaneamine, methylphenidate, cocaine, nikethamide, 3,4-methylenedioxyamphetamine, N-methyl-3,4-methylenedioxyamphetamine, strychnine, mesocarb, salbutamol, formoterol, clenbuterol, metandienone, stanozolol, bisoprolol, propranolol, metoprolol, anastrazole, clomiphene, exemestane, dexamethasone, budesonide, selective androgen receptor modulator (SARM) S4 (andarine), SARM S1, hydrochlorothiazide). Generally, only qualitative result interpretation was focussed upon, but for target analytes with deuterium-labelled internal standards (salbutamol, clenbuterol, cocaine, dexamethasone, THC-COOH and THC) quantitative analysis was also possible. Especially the most challenging analytes, THC and its carboxy metabolite, were detected in DBS at relevant concentrations (<0.5 ng/mL) using targeted HCD experiments. The method was validated for the parameters: specificity, linearity (0–20 ng/mL), precision (<25%), recovery (mean 60%), limit of detection/quantification, ion suppression, stability and accuracy (80–120%). Six isotope-labelled analogues used as internal standards facilitate a quantitative result interpretation which is of utmost importance especially for in-competition drug sports testing.

Keywords: Sports drug testing; QExactive; Unknown screening; Blood analysis

Site-specific protein glycosylation analysis with glycan isomer differentiation by Serenus Hua; Charles C. Nwosu; John S. Strum; Richard R. Seipert; Hyun Joo An; Angela M. Zivkovic; J. Bruce German; Carlito B. Lebrilla (pp. 1291-1302).

Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system. Here, we developed a novel method of comprehensive glycosite profiling using nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) that shows glycan isomer-specific differentiation on specific sites. Glycoproteins were digested by controlled non-specific proteolysis in order to produce informative glycopeptides. High-resolution, isomer-sensitive chromatographic separation of the glycopeptides was achieved using microfluidic chip-based capillaries packed with graphitized carbon. Integrated LC/MS/MS not only confirmed glycopeptide composition but also differentiated glycan and peptide isomers and yielded structural information on both the glycan and peptide moieties. Our analysis identified at least 13 distinct glycans (including isomers) corresponding to five compositions at the single N-glycosylation site on bovine ribonuclease B, 59 distinct glycans at five N-glycosylation sites on bovine lactoferrin, 13 distinct glycans at one N-glycosylation site on four subclasses of human immunoglobulin G, and 20 distinct glycans at five O-glycosylation sites on bovine κ-casein. Porous graphitized carbon provided effective separation of glycopeptide isomers. The integration of nano-LC with MS and MS/MS of non-specifically cleaved glycopeptides allows quantitative, isomer-sensitive, and site-specific glycoprotein analysis. Figure Overlaid chromatograms and associated structural assignments of glycopeptides from bovine κ-casein. Color denotes the site(s) of glycosylation from which the glycopeptide originated

Keywords: Site-specific glycosylation; Glycopeptide; Non-specific protease; Isomer; Quantitation; LC/MS

Binding interface of cardiac potassium channel proteins identified by hydrogen deuterium exchange of synthetic peptides by Jerri Chen; Ruth Angeletti; Thomas V. McDonald; Hui Xiao (pp. 1303-1309).

Three synthetic peptides, derived from the human potassium channel proteins Ether-a-go-go-related gene (HERG), KCNQ1, and KCNE1, were investigated by hydrogen deuterium exchange coupled with electron-transfer dissociation mass spectrometry at single residue resolution. Each amino acid residue in the first half of the HERG peptide incorporated deuterons with a higher rate than those in the second half of the peptide, consistent with the nuclear magnetic resonance structure of this peptide, with amino acids 1–10 being a flexible coil, whereas amino acids 11–24 are a stable amphipathic helix. The binding interface of KCNQ1 and KCNE1 was determined by comparing the difference of sequential fragment ions before and after binding. The residues determined to be involved in binding were consistent with a cysteine cross-linking study and confirmed by double mutant cycle analysis.

Keywords: Hydrogen deuterium exchange (HDX); Electron-transfer dissociation mass spectrometry; Deuterium incorporation; Hydrogen scrambling; c-Type fragment ions; z-Type fragment ions

A metallomics approach discovers selenium-containing proteins in selenium-enriched soybean by Qilin Chan; Joseph A. Caruso (pp. 1311-1321).

Our previous study found that high-molecular-weight selenium (Se) species make up 82% of the total Se in the bean of Se-enriched soybean plants (Chan et al. 2010, Metallomics, 2(2): p. 147–153). The Se species have been commonly seen in other plants in addition to soybean, but their identities remain unresolved. The present study employs a multi-technique metallomics approach to characterize the proteins containing Se in the beans of Se-enriched soybean plants. Two main categories of proteins, maturation proteins and protease inhibitors, were found in Se-containing high-performance liquid chromatography (HPLC) fractions. The proteins were screened by two-dimensional HPLC-inductively coupled plasma mass spectrometry, size-exclusion chromatography, and anion-exchange chromatography, and the Se-containing fractions were then identified by peptide mapping using HPLC-Chip-electrospray ion trap mass spectrometry. Based on the belief that Se goes into proteins through non-specific incorporation, a new method was designed and applied for the Se-containing peptide identification. The Se-containing peptide KSDQSSSYDDDEYSKPCCDLCMCTRS, part of the sequence of protein Bowman–Birk proteinase isoinhibitor (Glycine max), was found in one of the Se-containing fractions. The nutritional value of the Se-containing proteins in Se-enriched soybeans will be an interesting topic for the future studies.

Keywords: Selenium speciation; Metallomics; Soybean; ICP-MS; Selenium-containing proteins

Highly efficient precipitation of phosphoproteins using trivalent europium, terbium, and erbium ions by Yüksel Güzel; Matthias Rainer; Munazza Raza Mirza; Günther K. Bonn (pp. 1323-1331).

This study describes a highly efficient method for the selective precipitation of phosphoproteins by trivalent europium, terbium, and erbium metal ions. These metal cations belong to the group of lanthanides and are known to be hard acceptors with an overwhelming preference for oxygen-containing anions such as phosphates to which they form very tight ionic bonds. The method could be successfully applied to specifically precipitate phosphoproteins from complex samples including milk and egg white by forming solid metal–protein complexes. Owing to the low solubility product of the investigated lanthanide salts, the produced metal–protein complexes showed high stability. The protein pellets were extensively washed to remove nonphosphorylated proteins and contaminants. For the analysis of proteins the pellets were first dissolved in 30 % formic acid and subjected to matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) MS. For peptide mass-fingerprint analysis the precipitated phosphoproteins were enzymatically digested using microwave-assisted digestion. The method was found to be highly specific for the isolation and purification of phosphoproteins. Protein quantification was performed by colorimetric detection of total precipitated phosphoproteins and revealed more than 95 % protein recovery for each lanthanide salt.

Keywords: Europium; Terbium; Erbium; Phosphoproteomics; Precipitation; Enrichment

The analysis of major impurities of lipophilic-conjugated phosphorothioate oligonucleotides by ion-pair reversed-phase HPLC combined with MALDI-TOF-MS by Cai-Hong Liu; Dan-Dan Lu; Xin-Xiu Deng; Ying Wang; Jing-Yu Zhang; Yu-Lin Zhang; Sheng-Qi Wang (pp. 1333-1342).

A simple and rapid ion-pair reversed phase high-performance liquid chromatography (IP-RP-HPLC) method was developed to analyse the major impurities of lipophilic-conjugated phosphorothioate oligonucleotides (ODNs), which provided better separation performance than capillary gel electrophoresis and ion exchange chromatograph methods. The study showed that covalent conjugations of lipophilic group (docosanyl, C₂₂) to ODN at 5′-termini (denoted as 5′C₂₂-Flu) or 3′-termini (denoted as 3′C₂₂-Flu) exhibited similar chromatographic retention behavior. Some important analytical conditions of IP-RP-HPLC, including column type, ion-pairing buffer composition, and separation temperature, were investigated for the effects on the separation of crude 5′C₂₂-Flu. As expected, the method developed was successfully applied to the analysis of crude 3′C₂₂-Flu and both purified products. Furthermore, the related impurities derived from the synthetic process were identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrum. These MS results are of benefit to understanding the major process-related impurities in lipophilic-ODN conjugates synthesis, thereby elevating the quality of target products.

Keywords: Analysis of major impurities; Ion-pair reversed-phase HPLC; Lipophilic-conjugated phosphorothioate oligonucleotides; MALDL-TOF-MS

Au:CdHgTe quantum dots for in vivo tumor-targeted multispectral fluorescence imaging by Sihai Han; Ying Mu; Qiangyuan Zhu; Yibo Gao; Zuhong Li; Qinhan Jin; Wei Jin (pp. 1343-1352).

Near-infrared gold-doped CdHgTe quantum dots (QDs) with improved photoluminescence and biocompatibility were developed using an aqueous solution route with l-glutathione and l-cysteine as stabilizers. As-prepared Au:CdHgTe QDs were covalently linked to arginine–glycine–aspartic acid (RGD) peptide, anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MAb), and anti- carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) MAb separately. Three Au:CdHgTe QD bioconjugates (QD800-RGD, QD820-anti-CEACAM1, and QD840-anti-EGFR) were successfully used as probes for in vivo tumor-targeted multispectral fluorescence imaging of xenografts. Fluorescence signals from the QD bioconjugates used to detect three tumor markers were spectrally unmixed, and their co-localization was analyzed. The results indicate that multiple tumor markers could be simultaneously detected by multispectral fluorescence imaging in vivo using QD bioconjugates as probes. This approach has excellent potential as an imaging method for the noninvasive exploration and detection of multiple tumor markers in vivo, thereby substantially aiding the diagnosis of cancer. Figure In vivo tumor-targeted multispectral fluorescence imaging with Au:CdHgTe quantum dots

Keywords: Au:CdHgTe; Quantum dots; Tumor; Multispectral; Fluorescence; Imaging

Determination of trehalose-6-phosphate in Arabidopsis thaliana seedlings by hydrophilic-interaction liquid chromatography–mass spectrometry by Javier Sastre Toraño; Thierry L. Delatte; Henriette Schluepmann; Sjef C. M. Smeekens; Gerhardus J. de Jong; Govert W. Somsen (pp. 1353-1360).

A hydrophilic-interaction chromatography (HILIC) method coupled to electrospray ionization mass spectrometry (ESI-MS) was developed for the determination of trehalose-6-phophate (Tre6P) in Arabidopsis thaliana seedlings. The method was optimized for MS detection and separation of Tre6P from its isomers, such as sucrose-6-phosphate, by testing eluent pH, type of organic solvent and alkalinizer, and gradient conditions. Tre6P could be resolved from matrix components within 28 min by using a water–acetonitrile gradient (0.2 ml/min) at pH 12 with piperidine as alkalinizer. The method was validated for concentrations between 25 and 4,000 nM Tre6P in A. thaliana seedling extracts. Seedlings were extracted with consecutive liquid-liquid and solid-phase extractions, and analyzed with HILIC-MS. Obtained accuracy (80–120 %) and precision (<24 %) demonstrated the suitability of HILIC-MS for determining Tre6P level variations in plants. The limit of detection (LOD) was 3.5 nM Tre6P in extracts corresponding to 4.1 pmol.g⁻¹ fresh plant weight (FW). This is a considerable improvement with respect to anion-exchange chromatography (AEC)-MS (40 nM) and capillary electrophoresis-MS (80 nM). Furthermore, HILIC-MS analysis times were shorter than with AEC-MS (30 and 60 min, respectively). The applicability of the HILIC-MS method was demonstrated by the analysis of extracts from seedlings grown on medium containing 100 mM sorbitol or trehalose, resulting in mean Tre6P concentrations of 0.2 and 1.9 nmol.g⁻¹ FW, respectively. Similar concentrations were found with AEC-MS. HILIC-MS was also evaluated at a high flow rate (2.0 ml/min). This high-speed method resolved the Suc6P and Tre6P peaks within 3 min yielding a detection limit of 1.3 nM Tre6P. Figure Extracted-ion chromatogram obtained during high-pressure hydrophillic-interaction liquid chromatography-mass spectrometry of a trehalose-grown Arabidopsis thaliana (photopgraph) seedling. Trehalose-6-phosphate (structural formula) is separated from its isomers sucrose-6-phosphate and lactose-1-phosphate

Keywords: HILIC; Mass spectrometry; Carbohydrates; Trehalose-6-phosphate; Arabidopsis thaliana ; Phosphodisaccharides

Multiplexed immunoassay to detect anabolic androgenic steroids in human serum by Nuria Tort; J.-Pablo Salvador; M.-Pilar Marco (pp. 1361-1371).

A multianalyte enzyme-linked immunosorbent assay (ELISA) has been developed for the simultaneous detection of anabolic androgenic steroids (AAS) in human serum. The multiplexed method was developed according to a planar strategy in which the analytes are identified by their location in the microtiter plate. In the immunochemical procedure established here, human serum samples are mixed with a cocktail of antibodies and added to the distinct sections of a microplate biofunctionalized with different haptenized biomolecules. The cocktail of antibodies consists of a mixture of polyclonal antibodies raised against stanozolol (ST), boldenone (B), and tetrahydrogestrinone (THG). The whole immunochemical analytical procedure takes around 2 h including sample preparation, and many samples can be processed simultaneously to screen for the presence of the three AAS in a single run. Using this ELISA, ST, B, and THG can be detected and quantified individually. When used as a screening method, due to the cross-reactivity profiles of the immunoreagents used, the presence of up to 11 AAS can be detected simultaneously. The detectabilities achieved by this method in human serum are below the MRPLs (minimum required performance limits) proposed by WADA (World Anti-Doping Agency) and reference laboratories of the European Community. Figure Scheme of the multiplexed ELISA procedure.

Keywords: Anabolic-androgenic steroids (AAS); ELISA; Multianalyte; Human serum

A multiplexed screening method for agonists and antagonists of the estrogen receptor protein by Zhonghui Li; Ming Yan; Zhoumin Li; Maika Vuki; Dan Wu; Fei Liu; Wenying Zhong; Luyong Zhang; Danke Xu (pp. 1373-1384).

The estrogen receptor (ER) is regarded as a significant drug target because of its important physical and pathological function. In this article, we describe a novel screening method to obtain agonists and antagonists of ER. ER was immobilized onto an aldehyde-modified glass slide. The affinity of Cy3-labeled estradiol for ER protein microarrays was then determined. Two libraries, one containing 29 synthetic compounds and the other with 384 natural products that served as a model, were screened to find new ligands for ER. The IC₅₀ values obtained for tamoxifen and raloxifene were consistent with those found in the literature (4.85 × 10⁻⁷ M versus 1.74~4.23 × 10⁻⁷ M and 7.58 × 10⁻⁸ M versus 0.89~5.84 × 10⁻⁸ M, respectively). Finally, 65 active ligands (5 synthetic compounds and 60 natural products) of ER were identified. This novel method gave identical results to a conventional fluorescence polarization assay, thus verifying the accuracy of this simultaneous multireceptor screening method based on protein microarrays. The presented method is sensitive, accurate, and reliable, and shows great potential for use in high-throughput drug-screening research.

Keywords: Estrogen receptor; High-throughput screening; Protein–drug interaction; Protein microarray

Development of a simultaneous analytical method for selected anorectics, methamphetamine, MDMA, and their metabolites in hair using LC-MS/MS to prove anorectics abuse by Sooyeun Lee; Jihyun Kim; Sanghwan In; Hwakyung Choi; Seung Min Oh; Choon-Gon Jang; Kyu Hyuck Chung (pp. 1385-1394).

Owing to the tight control of methamphetamine, it is presumed that phentermine, an amphetamine-type anorectic, has recently been considered a supplement for methamphetamine abusers in Korea. In addition, the abuse of other anorectics obtained by inappropriate means has become a social issue. Hair is a useful specimen to prove chronic drug use. Therefore, an analytical method for the simultaneous detection of phentermine, phendimetrazine, amfepramone, fenfluramine, mazindol, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA), as well as their metabolites, which covers the major amphetamines and anorectic agents in Korea, in hair was established and validated using liquid chromatography–tandem mass spectrometry (LC-MS/MS). The drugs and their metabolites in hair were extracted using 1 % HCl in methanol and then filtered and analyzed by LC-MS/MS with electrospray ionization in positive mode. The validation results for selectivity, linearity, matrix effect, recovery, process efficiency, intra- and interassay precision and accuracy, and processed sample stability were satisfactory. The limits of detection ranged from 0.025 to 1 ng/10 mg hair and the limits of quantification were 0.25 ng/10 mg hair for every analyte except mazindol and phentermine, for which they were 10 ng/10 mg hair. The method was successfully applied for the segmental determination of selected anorectics, methamphetamine, MDMA, and their metabolites in hair from 39 drug suspects. Among the anorectics, phentermine and/or phendimetrazine were identified with or without methamphetamine in the hair samples. Closer supervision of the inappropriate use of anorectics is necessary. Also, hair analysis is useful for monitoring the abuse potential of unnoticed drugs.

Keywords: Drug abuse; Anorectics; Amphetamines; Hair analysis; Liquid chromatography–tandem mass spectrometry

Physicochemical properties of pH-controlled polyion complex (PIC) micelles of poly(acrylic acid)-based double hydrophilic block copolymers and various polyamines by J. Warnant; N. Marcotte; J. Reboul; G. Layrac; A. Aqil; C. Jerôme; D. A. Lerner; C. Gérardin (pp. 1395-1404).

The physicochemical properties of polyion complex (PIC) micelles were investigated in order to characterize the cores constituted of electrostatic complexes of two oppositely charged polyelectrolytes. The pH-sensitive micelles were obtained with double hydrophilic block copolymers containing a poly(acrylic acid) block linked to a modified poly(ethylene oxide) block and various polyamines (polylysine, linear and branched polyethyleneimine, polyvinylpyridine, and polyallylamine). The pH range of micellization in which both components are ionized was determined for each polyamine. The resulting PIC micelles were characterized using dynamic light scattering and small-angle X-ray scattering experiments (SAXS). The PIC micelles presented a core–corona nanostructure with variable polymer density contrasts between the core and the corona, as revealed by the analysis of the SAXS curves. It was shown that PIC micelle cores constituted by polyacrylate chains and polyamines were more or less dense depending on the nature of the polyamine. It was also determined that the density of the cores of the PIC micelles depended strongly on the nature of the polyamine. These homogeneous cores were surrounded by a large hairy corona of hydrated polyethylene oxide block chains. Auramine O (AO) was successfully entrapped in the PIC micelles, and its fluorescence properties were used to get more insight on the core properties. Fluorescence data confirmed that the cores of such micelles are quite compact and that their microviscosity depended on the nature of the polyamine. The results obtained on these core–shell micelles allow contemplating a wide range of applications in which the AO probe would be replaced by various cationic drugs or other similarly charged species to form drug nanocarriers or new functional nanodevices. Figure Poly ion complex micelles of PAA-b-PAMPEO double hydrophilic block copolymer and polyamines containing AO in the micelle core

Keywords: Hydrosoluble polymers; Polyion complex micelles; pH trigger; Fluorescence (AO); Core–shell nanoparticles; Scattering methods

Characterisation of historic plastics using terahertz time-domain spectroscopy and pulsed imaging by Gianluca Pastorelli; Tanja Trafela; Phillip F. Taday; Alessia Portieri; David Lowe; Kaori Fukunaga; Matija Strlič (pp. 1405-1414).

Terahertz (THz) time-domain spectroscopy and 3D THz pulsed imaging have been explored with regard to polymer materials, both commodity and historic polymers. A systematic spectroscopic study of a wide range of different polymer materials showed significant differences in their spectra. Polyolefins and polystyrenes generally exhibit lower absorption than other examined polymers, various cellulose derivates, poly(vinyl chloride), poly(methyl methacrylate), polyamide, hard rubber and phenol formaldehyde resin, the last of these exhibiting the most intense absorption over the entire range, 0.15–4.2 THz. It was also examined how the presence of plasticisers in poly(vinyl chloride), the presence of fillers in polypropylene, and the degree of branching in polyethylene and polystyrene affect the spectra; inorganic fillers in polypropylene affected the absorption most. With 3D THz pulsed imaging, features in polymer objects were explored, appearing either as integral parts of the material (coatings and pores in foams) or as a consequence of physical deterioration (cracks, delamination). All of these features of various complexities can be successfully imaged in 3D. Terahertz technology is thus shown to have significant potential for both chemical and structural characterisation of polymers, which will be of interest to heritage science, but also to the polymer industry and development of analytical technologies in general.

Keywords: Terahertz time-domain spectroscopy; Terahertz pulsed imaging; Polymers; Degradation; Heritage science

Chain-length-identification strategy in zinc polyphosphate glasses by means of XPS and ToF-SIMS by Maura Crobu; Antonella Rossi; Filippo Mangolini; Nicholas D. Spencer (pp. 1415-1432).

The surface chemistry of amorphous zinc polyphosphates of different compositions (ranging from zinc metaphosphate to zinc orthophosphate) has been investigated by means of X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary-ion mass spectroscopy (ToF-SIMS). The identification of the chain length of zinc polyphosphates by XPS was on the basis of the integrated intensity ratio of the bridging (P–O–P) and nonbridging (P = O and P–O–M) oxygen peaks used for fitting the oxygen 1s signal, the shift of the P 2p_3/2 signal towards lower binding energies and the modified Auger parameter towards higher values as the zinc content increases. The discrimination of the polyphosphate chain lengths was also achieved by ToF-SIMS, by comparing the intensities of selected characteristic phosphate fragments. Both techniques appear to be suitable for the investigation of polyphosphate glasses in applications such as tribology, where there is a need to identify the chain length present in the outermost monolayer of the film. Fourier-transform infrared (FT-IR) spectroscopy was used to characterize the bulk compounds. The FT-IR studies showed that long-chain structures linked through P–O–P bonds predominate in the metaphosphate composition, while when the zinc content is increased, the chains become shorter, ultimately being replaced by PO₄ monomers in the orthophosphate composition. Figure

Keywords: Polyphosphate glasses; X-ray photoelectron spectroscopy; Time-of-flight secondary-ion mass spectroscopy; Fourier transform IR spectroscopy

A rapid analytical method for cholecalciferol (vitamin D₃) in fortified infant formula, milk and milk powder using Diels–Alder derivatisation and liquid chromatography–tandem mass spectrometric detection by Grant A. Abernethy (pp. 1433-1440).

A method for analysing vitamin D₃ (VD3, cholecalciferol) has been established and validated. This method is rapid and cost effective and is intended for use in quality control in the manufacture of fortified infant formulae and milk powders. Milk or reconstituted milk powder was solubilised in methanol and extracted in one step into isooctane, which was separated by centrifugation. A portion of the isooctane layer was then transferred, and an aliquot of 4-phenyl-1,2,4-triazoline-3,5-dione was added to derivatise VD3. The analyte was then re-extracted into a small volume of acetonitrile and analysed by reverse-phase chromatography. Detection was by triple quadrupole mass spectrometer using a selective transition, m/z 560 → 298. An internal standard, deuterium-labelled VD3, was used to correct for losses in extraction and any variation in derivatisation and ionisation efficiencies. The method has been subjected to a single-laboratory validation and has been found to be linear, highly selective and accurate with respect to National Institute of Standards and Technology Standard Reference Material 1849, analyte spiking experiments and comparison with an LC–UV-based method. The repeatability standard deviation was 4.23 %. Significantly for routine laboratories, the method returns results within 2 h, generates minimal waste and minimises health and safety concerns to the analyst.

Keywords: Vitamin D₃ ; Cholecalciferol; Diels–Alder derivatisation; 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD); Infant formula; Milk

Fractionation of Sb and As in soil and sludge samples using different continuous-flow extraction techniques by E. Yu. Savonina; P. S. Fedotov; R. Wennrich (pp. 1441-1449).

The fractionation of Sb and As in soil and sludge samples had been comparably studied using two continuous-flow systems: a microcolumn (MC) and a rotating coiled column (RCC). The leachants were applied in correspondence with a five-step sequential extraction scheme addressing water-soluble, non-specifically sorbed, specifically sorbed, and bound to amorphous and crystalline Fe/Al oxide fractions of Sb and As. Inductively coupled plasma atomic emission spectroscopy was applied to determine antimony, arsenic, and major elements in the effluent and in the residual fractions after their digestion. Resemblances and discrepancies of the two methods were evaluated by the fractionation of Sb and As in forest soil, river sludge, and dumped waste (soil) samples. For the forest soil sample, which is very poor in organic matter, RCC and MC extractions yielded similar quantitative values of As and Sb contents in individual leachable fractions. However, for the river sludge sample with a moderate concentration of C _org (3.3 %), the results obtained by both continuous-flow methods are in satisfactory agreement. RCC extraction enabled water-soluble and non-specifically sorbed As fractions to be recovered, whereas after MC leaching, these environmentally relevant forms of As were not detected. For the soil rich in organic matter (C _org = 11.5 %), the discrepancy between the data of RCC and MC fractionations is significant. RCC extraction provides about six times higher recoveries of As and Sb bound to amorphous Fe/Al oxides. More efficient leaching of As and Sb in RCC may be attributed to the migration of organic-rich particles with low density inside the column that might enhance the mixing of the solid and liquid phases. Figure Dynamic fractionation approaches

Keywords: Soil; Dynamic fractionation; Rotating coiled column; Microcolumn; Sequential extraction; Arsenic; Antimony

Combination of sugar analysis and stable isotope ratio mass spectrometry to detect the use of artificial sugars in royal jelly production by Marine Wytrychowski; Gaëlle Daniele; Hervé Casabianca (pp. 1451-1456).

The effects of feeding bees artificial sugars and/or proteins on the sugar compositions and ¹³C isotopic measurements of royal jellies (RJs) were evaluated. The sugars fed to the bees were two C4 sugars (cane sugar and maize hydrolysate), two C3 sugars (sugar beet, cereal starch hydrolysate), and honey. The proteins fed to them were pollen, soybean, and yeast powder proteins. To evaluate the influence of the sugar and/or protein feeding over time, samples were collected during six consecutive harvests. ¹³C isotopic ratio measurements of natural RJs gave values of around −25 ‰, which were also seen for RJs obtained when the bees were fed honey or C3 sugars. However, the RJs obtained when the bees were fed cane sugar or corn hydrolysate (regardless of whether they were also fed proteins) gave values of up to −17 ‰. Sugar content analysis revealed that the composition of maltose, maltotriose, sucrose, and erlose varied significantly over time in accordance with the composition of the syrup fed to the bees. When corn and cereal starch hydrolysates were fed to the bees, the maltose and maltotriose contents of the RJs increased up to 5.0 and 1.3 %, respectively, compared to the levels seen in authentic samples (i.e., samples obtained when the bees were fed natural food: honey and pollen) that were inferior to 0.2% and not detected, respectively. The sucrose and erlose contents of natural RJs were around 0.2 %, whereas those in RJs obtained when the bees were fed cane or beet sugar were as much as 4.0 and 1.3 %, respectively. The combination of sugar analysis and ¹³C isotopic ratio measurements represents a very efficient analytical methodology for detecting (from early harvests onward) the use of C4 and C3 artificial sugars in the production of RJ.

Keywords: Royal jelly; IRMS; Sugar; Bee feeding; Gas chromatography

Erratum to: Development of a liquid chromatography–tandem mass spectrometry method for plasma-free metanephrines with ion-pairing turbulent flow online extraction by Xiang He; Marta Kozak (pp. 1457-1457).

Erratum to: Analytical methods for determination of new fluoroquinolones in biological matrices and pharmaceutical formulations by liquid chromatography: a review by Joana Sousa; Gilberto Alves; João Abrantes; Ana Fortuna; Amílcar Falcão (pp. 1459-1459).

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Analytical and Bioanalytical Chemistry (v.403, #5)