Analytical and Bioanalytical Chemistry (v.405, #26)

Multi-walled carbon nanotubes as sorptive material for solventless in-tube microextraction (ITEX2)—a factorial design study by Thorsten Hüffer; Xochitli L. Osorio; Maik A. Jochmann; Beat Schilling; Torsten C. Schmidt (8387-8395).
Multi-walled carbon nanotubes were evaluated as sorptive packing material for in-tube microextraction (ITEX2) in combination with GC-MS for the analysis of benzene, toluene, ethylbenzene, xylenes, and naphthalene in aqueous samples. For method development, a three-level full factorial design of experiment (DoE) was performed incorporating extraction temperature, number of extraction strokes, and extraction flow. The statistical analysis of method development showed that all considered extraction parameters significantly affected the extraction yield. Furthermore, it was shown that some factors significantly interacted with each other, which indicates the advantage of using DoE for method development. The thereby optimized ITEX2 protocol was validated regarding its linear dynamic range, method detection limit (MDL), and precision. The MDLs of investigated analytes ranged between 2 ng L−1 for naphthalene and 11 ng L−1 for p-xylene. The relatively low MDL obtained for naphthalene, despite its comparably low air–water partitioning, can be explained by its strong interaction with carbon nanotubes. All obtained MDLs are at least comparable to previous reports on microextraction techniques, emphasizing both the quality of ITEX2 and the highly promising sorbent characteristics of carbon nanotubes. Furthermore, the method was applied to three real samples, which demonstrated good recoveries of analytes from tap water, a bank filtrate, and an effluent from a wastewater treatment plant. Figure MWCNTs as sorptive material for ITEX2
Keywords: Carbon nanotubes; In-tube microextraction; Design of experiment; GC-MS; VOCs; Water samples

The combined use of sorbent tubes (ST) and thermal desorption (TD) has become the common practice for the trace-level analysis of gaseous volatile organic compounds (VOCs). In this research, the potential bias in VOC analysis due to the solvent introduced into the system as a liquid standard (LS) is examined in three stages by analyzing LSs of 19 VOCs in methanol solvent against a three-bed ST (Tenax TA, Carbopack B, and Carboxen 1000). In experimental stage 1, LS made at four concentration levels (between 10 and 150 ng μL−1) were each analyzed at four injection volumes (1, 2, 5, and 10 μL) based on a vaporization method. In experimental stage 2, calibration was also conducted by direct injection over an extended concentration range at two volumes, 1 and 10 μL. In experimental stage 3, the response factors (RF) of a single analyte mass were compared across the four injection volumes and between two injection methods. These results were analyzed to explore the complex relationship between variables such as LS volume, target/solvent chemical type, sorbent strength, and prepurge condition. There was no change in the ST/TD performance up to 2 μL of LS. However, as the injection volume increased up to 5 μL, a notable shift in RF and retention time occurred (e.g., for benzene and methyl ethyl ketone). At the maximum injection volume (10 μL), a significant reduction in sensitivity is evident for all compounds, e.g., 50 % drops relative to 1 μL injection. As such, the TD performance tends to deteriorate with increasing volume of methanol initially loaded on the ST. Although the dominant fraction of solvent was removed by two prepurge steps, residue caught in the strong sorbent fraction is still found to exert an effect on the subsequent analysis, e.g., delayed retention, sensitivity reduction, or disappearance of certain compounds.
Keywords: Thermal desorption; Bias; Calibration; Matrix effect; Methanol; Prepurge; VOC

VIZR—an automated chemometric technique for metabolic profiling by Gregory A. Barding Jr.; Daniel J. Orr; Sumukh M. Sathnur; Cynthia K. Larive (8409-8417).
A chemometric technique, visual interpretation of z-score ratios (VIZR), written in the open source code R, has been developed to identify metabolic differences between individual biosamples and a control group. To demonstrate the capabilities of VIZR, 49 urine samples were collected from healthy volunteers: 41 samples were collected randomly following a normal dietary routine and 7 test samples were collected after dietary supplementation with either ibuprofen or alcoholic beverages. An eighth test sample was prepared by 50 % dilution of a control sample. Sample analysis was conducted by 1H nuclear magnetic resonance (NMR) spectroscopy and the collected data were subjected to VIZR analysis, which successfully discriminated each of the 8 test samples from the 41 control samples. In addition, VIZR analysis revealed the NMR spectral regions responsible for the disparity between the individual test samples and the control group. The self-normalizing nature of the VIZR calculation provides a robust analysis independent of dilution effects, which is especially important in urine analyses. Potential applications of VIZR include high-throughput data analysis for toxicological profiling, disease diagnosis, and biomarker identification in any type of biosample for which a control dataset can be established. Although demonstrated herein for the statistical analysis of 1H NMR data, the VIZR program is platform independent and could be applied to digitized metabolic datasets acquired using other techniques including hyphenated mass spectrometry measurements. Figure The VIZR z-score scatter plot identifies the test samples that differ from the controls
Keywords: Chemometrics; NMR spectroscopy; Biomarker detection; Toxicological profiling; Disease detection; Z-score; Metabolomics; Metabonomics

Nuclear magnetic resonance (NMR) spectroscopy-based metabonomics is of growing importance for discovery of human disease biomarkers. Identification and validation of disease biomarkers using statistical significance analysis (SSA) is critical for translation to clinical practice. SSA is performed by assessing a null hypothesis test using a derivative of the Student's t test, e.g., a Welch's t test. Choosing how to correct the significance level for rejecting null hypotheses in the case of multiple testing to maintain a constant family-wise type I error rate is a common problem in such tests. The multiple testing problem arises because the likelihood of falsely rejecting the null hypothesis, i.e., a false positive, grows as the number of tests applied to the same data set increases. Several methods have been introduced to address this problem. Bonferroni correction (BC) assumes all variables are independent and therefore sacrifices sensitivity for detecting true positives in partially dependent data sets. False discovery rate (FDR) methods are more sensitive than BC but uniformly ascribe highest stringency to lowest p value variables. Here, we introduce standard deviation step down (SDSD), which is more sensitive and appropriate than BC for partially dependent data sets. Sensitivity and type I error rate of SDSD can be adjusted based on the degree of variable dependency. SDSD generates fundamentally different profiles of critical p values compared with FDR methods potentially leading to reduced type II error rates. SDSD is increasingly sensitive for more concentrated metabolites. SDSD is demonstrated using NMR-based metabonomics data collected on three different breast cancer cell line extracts.
Keywords: Bioanalytical methods; Bioassays; Biological samples; Chemometrics/statistics; NMR/ESR

Optimised protocols for the metabolic profiling of S. cerevisiae by 1H-NMR and HRMAS spectroscopy by Martina Palomino-Schätzlein; Maria Micaela Molina-Navarro; Marta Tormos-Pérez; Susana Rodríguez-Navarro; Antonio Pineda-Lucena (8431-8441).
An optimised extraction protocol for the analysis of Saccharomyces cerevisiae aqueous and organic metabolites by nuclear magnetic resonance spectroscopy that allows the identification and quantification of up to 50 different compounds is presented. The method was compared with other metabolic profiling protocols for S. cerevisiae, where generally different analytical techniques are applied for metabolite quantification. In addition, the analysis of intact S. cerevisiae cells by HRMAS was implemented for the first time as a complementary method. The optimised protocols were applied to study the metabolic effect of glucose and galactose on S. cerevisiae growth. Furthermore, the metabolic reaction of S. cerevisiae to osmotic stress has been studied. Figure Graphical description of the metabolic profiling study carried out on S. cerevisiae
Keywords: NMR; Yeast; HRMAS; Metabolomics; Stress

Studies on drug metabolism by fungi colonizing decomposing human cadavers. Part I: DNA sequence-based identification of fungi isolated from postmortem material by Jorge A. Martínez-Ramírez; Juliane Strien; Juliane Sanft; Gita Mall; Grit Walther; Frank T. Peters (8443-8450).
Cadavers can be colonized by a wide variety of bacteria and fungi. Some of these microbes could change the concentration or the metabolic pattern of drugs present in postmortem samples. The purpose of this study was to identify fungi from human postmortem material and to further assess their potential role in the metabolism of drugs. Aliquots of 252 postmortem samples (heart blood, liver, kidney, and lung) taken from 105 moderately to severely decomposed bodies were streaked on Sabouraud agar for isolation of fungal species. One part of the samples was worked up immediately after autopsy (group I). The second part had previously been stored at −20 °C for at least 1 year (group II). Identification of the isolates was achieved morphologically by microscopy and molecularly by polymerase chain reaction amplification and sequencing of markers allowing species identification of the respective genera. Depending on the genus, different gene fragments were used: calmodulin for Aspergillus, β-tubulin for Penicillium, translation elongation factor 1α for Fusarium, and the internal transcribed spacer region of the ribosomal DNA for all remaining genera. A total of 156 fungal strains were isolated from 62 % of the postmortem materials. By using these primers, 98 % of the isolates could be identified to the species level. The most common genera were Candida (60.0 %—six species), Penicillium (10.3 %—two species), Rhodotorula (7.1 %—one species), Mucor (6.4 %—four species), Aspergillus (3.2 %—four species), Trichosporon (3.2 %—one species), and Geotrichum (3.2 %—one species). Group I samples contained 53 % more fungal species than stored samples suggesting some fungi did not survive the freezing process. The isolated fungi might be characteristic for decomposed bodies. The proposed methodology proved to be appropriate for the identification of fungi in this type of material.
Keywords: Fungi; Cadavers; Postmortem material; PCR; Molecular identification

Oral fluid cannabinoid concentrations following controlled smoked cannabis in chronic frequent and occasional smokers by Sebastien Anizan; Garry Milman; Nathalie Desrosiers; Allan J. Barnes; David A. Gorelick; Marilyn A. Huestis (8451-8461).
Oral fluid (OF) is an alternative biological matrix for monitoring cannabis intake in drug testing, and drugged driving (DUID) programs, but OF cannabinoid test interpretation is challenging. Controlled cannabinoid administration studies provide a scientific database for interpreting cannabinoid OF tests. We compared differences in OF cannabinoid concentrations from 19 h before to 30 h after smoking a 6.8 % THC cigarette in chronic frequent and occasional cannabis smokers. OF was collected with the Statsure Saliva Sampler™ OF device. 2D-GC-MS was used to quantify cannabinoids in 357 OF specimens; 65 had inadequate OF volume within 3 h after smoking. All OF specimens were THC-positive for up to 13.5 h after smoking, without significant differences between frequent and occasional smokers over 30 h. Cannabidiol (CBD) and cannabinol (CBN) had short median last detection times (2.5–4 h for CBD and 6–8 h for CBN) in both groups. THCCOOH was detected in 25 and 212 occasional and frequent smokers’ OF samples, respectively. THCCOOH provided longer detection windows than THC in all frequent smokers. As THCCOOH is not present in cannabis smoke, its presence in OF minimizes the potential for false positive results from passive environmental smoke exposure, and can identify oral THC ingestion, while OF THC cannot. THC ≥ 1 μg/L, in addition to CBD ≥ 1 μg/L or CBN ≥ 1 μg/L suggested recent cannabis intake (≤13.5 h), important for DUID cases, whereas THC ≥ 1 μg/L or THC ≥ 2 μg/L cutoffs had longer detection windows (≥30 h), important for workplace testing. THCCOOH windows of detection for chronic, frequent cannabis smokers extended beyond 30 h, while they were shorter (0–24 h) for occasional cannabis smokers.
Keywords: Tetrahydrocannabinol; Cannabinoids; 11-Nor-9-carboxy-tetrahydrocannabinol; Oral fluid; Statsure Saliva Sampler; Drug testing

The continuing emergence of designer drugs imposes high demands on the scope and sensitivity of toxicological drug screening procedures. An ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) method was developed for screening and simultaneous confirmation of both designer drugs and other drugs of abuse in urine samples in a single run. The method covered selected synthetic cannabinoids and cathinones, amphetamines, natural cannabinoids, opioids, cocaine and other important drugs of abuse, together with their main urinary metabolites. The database consisted of 277 compounds with molecular formula and exact monoisotopic mass; retention time was included for 192 compounds, and primary and secondary qualifier ion exact mass for 191 and 95 compounds, respectively. Following a solid-phase extraction, separation was performed by UHPLC and mass analysis by HR-TOFMS. MS, and broad-band collision-induced dissociation data were acquired at m/z range 50–700. Compound identification was based on a reverse database search with acceptance criteria for retention time, precursor ion mass accuracy, isotopic pattern and abundance of qualifier ions. Mass resolving power in spiked urine samples was on average FWHM 23,500 and mass accuracy 0.3 mDa. The mean and median cut-off concentrations determined for 75 compounds were 4.2 and 1 ng/mL, respectively. The range of cut-off concentrations for synthetic cannabinoids was 0.2–60 ng/mL and for cathinones 0.7–15 ng/mL. The method proved to combine high sensitivity and a wide scope in a manner not previously reported in drugs of abuse screening. The method’s feasibility was demonstrated with 50 authentic urine samples. Figure Extracted ion chromatograms of metabolites of synthetic cannabinoids and their fragments, including a new common metabolite: JWH-072-propanoic acid
Keywords: Designer drug; Drugs of abuse; Urine; Toxicological screening; High-resolution mass spectrometry

Multiclass method for antimicrobial analysis in animal feeds by liquid chromatography–tandem mass spectrometry by S. Borràs; R. Companyó; J. Guiteras; J. Bosch; M. Medina; S. Termes (8475-8486).
A rapid multiclass method that covers 50 antimicrobials from 13 different families in animal feeds was developed. Samples were extracted using a mixture of methanol, acetonitrile and a McIlvaine buffer combined with sonication. Feed extracts were simply diluted prior to injection, since the clean-up strategies that were tested, based on either solid-phase extraction or dispersive solid-phase extraction, were ineffective at minimizing matrix-related signal suppression/enhancement. Analysis was carried out by liquid chromatography coupled to tandem mass spectrometry using an electrospray ionization source operating in positive and negative modes. For the quantification, matrix-fortified standard calibration curves were used to compensate for matrix effects and losses in sample preparation. The method was validated in-house in pig, poultry and cattle feed matrices and showed satisfactory performance characteristics. Thus, the proposed approach was suitable for application in a routine high-throughput laboratory for the official control of feeds. Figure Multiclass method for antimicrobial analysis in animal feeds.
Keywords: Animal feed; Antibiotics; Coccidiostats; Cross-contamination; Matrix effects; LC-MS/MS

Bioappearance and pharmacokinetics of bioactives upon coffee consumption by Roman Lang; Natalie Dieminger; Anja Beusch; Yu-Mi Lee; Andreas Dunkel; Barbara Suess; Thomas Skurk; Anika Wahl; Hans Hauner; Thomas Hofmann (8487-8503).
Habitual consumption of medium amounts of coffee over the whole life-span is hypothesized to reduce the risk to develop diabetes type 2 (DM2) and Alzheimer’s disease (AD). To identify putative bioactive coffee-derived metabolites, first, pooled urine from coffee drinkers and non-coffee drinkers were screened by UPLC-HDMS. After statistical data analysis, trigonelline, dimethylxanthines and monomethylxanthines, and ferulic acid conjugates were identified as the major metabolites found after coffee consumption. For quantitative analysis of these markers in body fluids, targeted methods based on stable-isotope dilution and UPLC-MS/MS were developed and applied to plasma samples from a coffee intervention study (n = 13 volunteers) who consumed a single cup of caffeinated coffee brew after a 10-day washout period. Chlorogenic acid-derived metabolites were found to be separated into two groups showing different pharmacokinetic properties. The first group comprised, e.g., ferulic acid and feruloyl sulfate and showed early appearance in the plasma (∼1 h). The second group contained particularly chlorogenic acid metabolites formed by the intestinal microflora, appearing late and persisting in the plasma (>6 h). Trigonelline appeared early but persisted with calculated half-life times ∼5 h. The plasma levels of caffeine metabolites significantly and progressively increased 2–4 h after coffee consumption and did not reach c max within the time frame of the study. The pharmacokinetic profiles suggest that particularly trigonelline, caffeine, its metabolites, as well as late appearing dihydroferulic acid, feruloylglycine and dihydroferulic acid sulfate formed from chlorogenic acid by the intestinal microflora accumulate in the plasma due to their long half-life times during habitual consumption of several cups of coffee distributed over the day. Since some of these metabolites have been reported to show antioxidant effects in vivo, antioxidant-response-element activating potential, and neuroprotective properties, respectively, some of these key metabolites might account for the inflammation- and DM2/AD risk reducing effects reported for habitual life time consumption of coffee. Figure Identification of coffee key metabolites in human urine using untargeted MS-based profiling and subsequent targeted analyses in plasma using stable isotope dilution assays
Keywords: UPLC-HDMS; Coffee; Chlorogenic acids; Trigonelline; Caffeine; Bioappearance

Top-down analysis of 30–80 kDa proteins by electron transfer dissociation time-of-flight mass spectrometry by Luca Fornelli; Julien Parra; Ralf Hartmer; Carsten Stoermer; Markus Lubeck; Yury O. Tsybin (8505-8514).
Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth structure characterization of large peptides, small- and medium-sized proteins, and non-covalent protein complexes. Here, we describe the performance of this approach for structural analysis of intact proteins as large as the 80 kDa serotransferrin. Current time-of-flight (TOF) MS technologies ensure adequate resolution and mass accuracy to simultaneously analyze intact 30–80 kDa protein ions and the complex mixture of their ETD product ions. Here, we show that ETD TOF MS is efficient and may provide extensive sequence information for unfolded and highly charged (around 1 charge/kDa) proteins of ∼30 kDa and structural motifs embedded in larger proteins. Sequence regions protected by disulfide bonds within intact non-reduced proteins oftentimes remain uncharacterized due to the low efficiency of their fragmentation by ETD. For serotransferrin, reduction of S–S bonds leads to significantly varied ETD fragmentation pattern with higher sequence coverage of N- and C-terminal regions, providing a complementary structural information to top-down analysis of its oxidized form. Figure ETD TOF MS provides extensive sequence information for unfolded and highly charged proteins of ~30 kDa and above. In addition to charge number and distribution along the protein, disulfide bonds direct ETD fragmentation. For intact non-reduced 80 kDa serotransferrin, sequence regions protected by disulfide bonds oftentimes remain uncharacterized. Reduction of disulfide bonds of serotransferrin increases ETD sequence coverage of its N- and C-terminal regions, providing a complementary structural information to the top-down analysis of its oxidized form
Keywords: Electron transfer dissociation; ETD; Electrospray; ESI; Tandem MS; MS/MS; Time-of-flight mass spectrometry; TOF MS; Top-down; Transferrin

Total synchronous fluorescence spectroscopic data modeled with first- and second-order algorithms for the determination of doxorubicin in human plasma by Agustina V. Schenone; María J. Culzoni; Andres D. Campiglia; Héctor C. Goicoechea (8515-8523).
In this work, we present the development of a method for the determination of doxorubicin in plasma samples in the presence of an unexpected component (riboflavin) by using total synchronous fluorescence spectroscopic data matrices. To the best of our knowledge, this is the first time that the second-order advantage is obtained with this kind of data. Two strategies including unfolding the data and: (a) processing with multivariate curve resolution coupled to alternating least-squares as first-order data or (b) processing with unfolded partial least-squares and exploiting the second-order advantage by the residual bilinearization procedure were considered. The calibration set was built with human plasma samples spiked with doxorubicin, while the validation set was prepared with human plasma samples spiked with both doxorubicin and riboflavin, a drug whose spectrum highly overlaps with the one corresponding to doxorubicin. Both methodologies reached good indicators of accuracy: recoveries of ca. 100 ± 8 % and REP of ca. 5 %; and precision: coefficient of variations between 7 and 9 %. Figure ᅟ
Keywords: Total synchronous fluorescence spectroscopy; MCR-ALS; UPLS/RBL; Doxorubicin

Time-resolved fluorescence microscopy for quantitative Ca2+ imaging in living cells by Kristina Sagolla; Hans-Gerd Löhmannsröben; Carsten Hille (8525-8537).
Calcium (Ca2+) is a ubiquitous intracellular second messenger and involved in a plethora of cellular processes. Thus, quantification of the intracellular Ca2+ concentration ([Ca2+]i) and of its dynamics is required for a comprehensive understanding of physiological processes and potential dysfunctions. A powerful approach for studying [Ca2+]i is the use of fluorescent Ca2+ indicators. In addition to the fluorescence intensity as a common recording parameter, the fluorescence lifetime imaging microscopy (FLIM) technique provides access to the fluorescence decay time of the indicator dye. The nanosecond lifetime is mostly independent of variations in dye concentration, allowing more reliable quantification of ion concentrations in biological preparations. In this study, the feasibility of the fluorescent Ca2+ indicator Oregon Green Bapta-1 (OGB-1) for two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was evaluated. In aqueous solution, OGB-1 displayed a Ca2+-dependent biexponential fluorescence decay behaviour, indicating the presence of a Ca2+-free and Ca2+-bound dye form. After sufficient dye loading into living cells, an in situ calibration procedure has also unravelled the Ca2+-free and Ca2+-bound dye forms from a global biexponential fluorescence decay analysis, although the dye's Ca2+ sensitivity is reduced. Nevertheless, quantitative [Ca2+]i recordings and its stimulus-induced changes in salivary gland cells could be performed successfully. These results suggest that OGB-1 is suitable for 2P-FLIM measurements, which can gain access to cellular physiology. Figure Procedure of quantitative Ca2+ recordings in cockroach salivary gland cells using time-resolved fluorescence measurements with the Ca2+-sensor OGB-1
Keywords: Fluorescence lifetime; TCSPC; Two-photon excitation; 2P cross section; Epithelial ion transport; OGB-1

Quantitative turbidity assay for lipolytic enzymes in microtiter plates by Susann Barig; Manja Schiemann; Vladimir M. Mirsky; K. Peter Stahmann (8539-8547).
A clearing assay for lipolytic enzymes has been realized in 96-well microtiter plates. A thin layer containing emulsified tributyrin as turbidity-generating substrate was placed on a thicker supporting aqueous layer. Both layers were stabilized by a gel-forming agent. Enzyme addition leads to clearing of the emulsion detected with a standard microtiter plate reader as a decrease of extinction. Dependencies of the signal kinetics on the substrate and enzyme concentrations were studied. For 0.5–1 % tributyrin content the reaction rate is not substrate-limited. An initial slope of the signal kinetics is proportional to the lipase activity. A detailed characterization of the assay was performed. Lipolysis of tributyrin was confirmed by glycerol detection. Various gel-forming agents were compared and diffusion conditions in these gels were analyzed. Agar and agarose were found to be the most suitable gel-forming agents, which do not affect enzyme diffusion whereas polyacrylamide gels block lipase diffusion and therefore are not suitable for the assay. The optimized assay prepared from 1 % tributyrin emulsion in 2 % agar gel was tested with six microbial lipases and porcine pancreatic lipase. The detection limit is 20–60 ng/well which is equivalent to 30 μU/well for T. lanuginosus lipase. Figure A clearing assay for lipolytic enzymes has been realized in 96-well microtiter plates. A thin layer containing emulsified tributyrin as turbidity-generating substrate was placed on a thicker supporting aqueous layer. Both layers are stabilized by a gel-forming agent. Enzyme addition leads to clearing of the emulsion detected with a standard microtiter plate reader as a decrease of extinction
Keywords: Lipase assay; Lipolytic enzymes; Microtiter plates; Turbidity; Tributyrin

Automated quantitative analysis of lipid accumulation and hydrolysis in living macrophages with label-free imaging by Wei-Wen Chen; Chen-Hao Chien; Chiung-Lin Wang; Huai-Hsien Wang; Yuh-Lin Wang; Shih-Torng Ding; Tzong-Shyuan Lee; Ta-Chau Chang (8549-8559).
The accumulation of lipids in macrophages is a key factor that promotes the formation of atherosclerotic lesions. Several methods such as biochemical assays and neutral lipid staining have been used for the detection of lipids in cells. However, a method for real-time quantitative assessment of the lipid content in living macrophages has yet to be shown, particularly for its kinetic process with drugs, due to the lack of suitable tools for non-invasive chemical detection. Here we demonstrate label-free real-time monitoring of lipid droplets (LDs) in living macrophages by using coherent anti-Stokes Raman scattering (CARS) microscopy. In addition, we have established an automated image analysis method based on maximum entropy thresholding (MET) to quantify the cellular lipid content. The result of CARS image analysis shows a good correlation (R 2 > 0.9) with the measurement of biochemical assay. Using this method, we monitored the processes of lipid accumulation and hydrolysis in macrophages. We further characterized the effect of a lipid hydrolysis inhibitor (diethylumbelliferyl phosphate, DEUP) and determined the kinetic parameters such as the inhibition constant, K i. Our work demonstrates that the automated quantitative analysis method is useful for the studies of cellular lipid metabolism and has potential for preclinical high-throughput screening of therapeutic agents related to atherosclerosis and lipid-associated disorders. Figure Automated quantitative analysis for the label-free detection of lipid content in living cells
Keywords: Image-based analysis; Macrophages; Lipid droplet; Lipid hydrolysis; Drug screening; Coherent anti-Stokes Raman scattering (CARS) microscopy

Integrins are ubiquitous adhesion receptors that are important for signaling and integrating the extracellular matrix and cytoskeleton. The role of cytoplasmic proteins vinculin, focal adhesion kinase (FAK), integrin-linked kinase (ILK), and membrane proteins epidermal growth factor receptor (EGFR) and Notch in altering αPS2CβPS integrin lateral diffusion was measured using single particle tracking (SPT) and RNA interference (RNAi). SPT measures heterogeneous diffusion properties, and RNAi selectively reduces the concentration of a target protein. After systematically reducing the concentration of vinculin, FAK, ILK, EGFR, or Notch, there was a 31 to 80 % increase in the mobile integrin fraction, indicating that these five targeted proteins (or assemblies that contain these proteins) are responsible for immobilizing a fraction of the integrins when all proteins are present at native concentrations. The average diffusion coefficient of all mobile integrins did not change after any of the RNAi treatments, and the percentage of Brownian, directed, or anomalous/constrained trajectories relative to total mobile trajectories did not change after vinculin or EGFR RNAi. However, the fraction of anomalous/constrained trajectories relative to the total mobile trajectories increased 9 to 19 % after FAK, ILK, and Notch RNAi, when the concentration of these proteins was reduced. In the case of FAK, ILK, and Notch, native concentrations of these proteins simultaneously increase the immobile fraction of integrins but decrease the diffusion constraints to those integrins that remain mobile. Comparisons of single receptor and ensemble measurements of diffusion and what is known about the effect of these proteins in altering integrin clustering are discussed. Figure Membrane and Cytoplasmic proteins that affect the heterogeneous diffusion properties of integrin receptors can be elucidated using RNA interference to reduce the concentration of a single protein combined with single particle tracking measurements at native or reduced protein concentrations. Trajectories exhibit directed, Brownian, or anomalous motion; there is also a fraction of immobile receptor.
Keywords: Single particle tracking; RNAi; αPS2CβPS integrins; S2 cells; Tiggrin-functionalized quantum dot

Sensitive routine liquid chromatography–tandem mass spectrometry method for serum estradiol and estrone without derivatization by Steven Pauwels; Leen Antonio; Ivo Jans; Anneleen Lintermans; Patrick Neven; Frank Claessens; Brigitte Decallonne; Jaak Billen; Dirk Vanderschueren; Pieter Vermeersch (8569-8577).
The need for a routinely applicable assay to measure low estradiol levels in adult men, postmenopausal women, and young adolescents was recently discussed in an Endocrine Society position statement. Our aim was to develop a sensitive liquid chromatography–tandem mass spectrometry method for estradiol and estrone in human serum without the need for derivatization or extended extraction protocols. After protein precipitation of serum with a mixture of methanol/acetonitrile (85/15) (v/v) containing isotopic internal standards (17β-estradiol-16,16,17-d 3 and estrone-2,3,4-13C), we quantified estradiol and estrone by two-dimensional liquid chromatography–tandem mass spectrometry with electrospray ionization in the negative mode monitoring 5 × 271.20→145.00 (17β-estradiol) and 269.20→145.00 (estrone). Sensitivity was increased by using fluoride and summation of 5 identical transitions for estradiol. Our method was analytically validated, compared against direct immunoassays using serum of 25 adult men, and clinically tested by measuring samples of 3 men at baseline and after chemical castration, 30 postmenopausal women and 15 patients receiving aromatase inhibitors. Total imprecision was below 20 % for the low quality controls. Limit of quantification was 1.3 ng/L (4.8 pmol/L) for estradiol and 1.2 ng/L (4.4 pmol/L) for estrone. Estradiol in Certified Reference Material BCR-576 was within specified uncertainty limits. No significant ion suppression or interference was observed. Our method showed modest correlation with direct immunoassay for estradiol (r 2 = 0.64) but no correlation for estrone (r 2 = 0.12). Patient sample results were within expected ranges. In conclusion, we developed a routinely applicable liquid chromatography–tandem mass spectrometry method for estradiol and estrone measurement which is sensitive enough for use in men, postmenopausal women, and young adolescents. Figure Chromatogram of E2 in serum with S/N for one MRM and for the summation of 5 identical MRMs
Keywords: Estradiol; Estrone; Mass spectrometry; Liquid chromatography; MRM summation

Separation and analysis of cis-diol-containing compounds by boronate affinity-assisted micellar electrokinetic chromatography by Heye Wang; Chenchen Lü; Hengye Li; Yang Chen; Min Zhou; Jian Ouyang; Zhen Liu (8579-8586).
Cis-diol-containing compounds (CDCCs) are usually highly hydrophilic compounds and are therefore difficult to separate by conventional reversed-phase-based micellar electrokinetic chromatography (MEKC) due to poor selectivity. Here, we report a new method, called boronate affinity-assisted micellar electrokinetic chromatography (BAA-MEKC), to solve this issue. A boronic acid with a hydrophobic alkyl chain was added to the background electrolyte, which acted as a modifier to adjust the selectivity. CDCCs can covalently react with the boronic acid to form negatively charged surfactant-like complexes, which can partition into micelles formed with a cationic surfactant. Thus, CDCCs can be separated according to the differential partition constants of their boronic acid complexes between the micellar phase and the surrounding aqueous phase. To verify this method, eight nucleosides were employed as the test compounds and their separation confirmed that the combination of boronate affinity interaction with MEKC can effectively enhance the separation of CDCCs. The effects of experimental conditions on the separation were investigated. Finally, the BAA-MEKC method was applied to the separation and analysis of nucleosides extracted from human urine. BAA-MEKC exhibited better selectivity and improved separation as compared with conventional MEKC and CZE. Successful quantitative analysis of urinary nucleosides by BAA-MEKC was demonstrated.
Keywords: Boronate affinity; Boronic acid; Cis-diol-containing compounds; Micellar electrokinetic chromatography; Selectivity; Separation

Novel moving reaction boundary-induced stacking and separation of human hemoglobins in slab polyacrylamide gel electrophoresis by Yun-Yun Tang; Hou-Yu Wang; Lu Chen; Si Li; Chen-Gang Guo; Hui-Zhi Fan; Cheng-Xi Cao; Liu-Yin Fan (8587-8595).
We developed a novel polyacrylamide gel electrophoresis (PAGE) method to stack and separate human hemoglobins (Hbs) based on the concept of moving reaction boundary (MRB). This differs from the classic isotachophoresis (ITP)-based stacking PAGE in the aspect of buffer composition, including the electrode buffer (pH 8.62 Tris–Gly), sample buffer (pH 6.78 Tris–Gly), and separation buffer (pH 8.52 Tris–Gly). In the MRB-PAGE system, a transient MRB was formed between alkaline electrode buffer and acidic sample buffer, being designed to move toward the anode. Hbs carried partial positive charges in the sample buffer due to its pH below pI values of Hbs, resulting in electromigrating to the cathode. Hbs would carry negative charges quickly when migrated into the alkaline electrode buffer and be transported to the anode until meeting the sample buffer again. Thus, Hbs were stacked within a MRB until the transient MRB reached the separation buffer and then separated by zone electrophoresis with molecular sieve effect of the gel. The experimental results demonstrated that there were three clear and sharp protein zones of Hbs (HbA1c, HbA0, and HbA2) in MRB-PAGE, in contrast to only one protein zone (HbA0) in ITP-PAGE for large-volume loading (≥15 μl), indicating high stacking efficiency, separation resolution, and good sensitivity of MRB-PAGE. In addition, MRB-PAGE was performed in a conventional slab PAGE device, requiring no special device. Thus, it could be widely used in separation and analysis of diluted protein in a standard laboratory. Figure Diagram of MRB-induced stacking in a slab PAGE. (A) arrangement of separation buffer (pH 8.01–9.55 Tris–Gly), sample buffer (pH 6.37–7.22 Tris–Gly), and electrode buffer (pH 8.21–9.05 Tris–Gly); (B) initial MRB formed between electrode and sample buffers for stacking of low-content Hbs in sample buffer under electric field; (C) MRB moving toward the anode and partly stacking of Hbs within the MRB; (D) quasi-complete stacking of Hbs via MRB closing to the separating gel; (E) separation of Hbs in a zone electrophoresis mode
Keywords: Moving reaction boundary; Isotachophoresis; Stacking; Polyacrylamide gel electrophoresis; Human hemoglobins

To obtain fast separation, ionic liquids were used as porogens first in combination with reversible addition–fragmentation chain transfer (RAFT) polymerization to prepare a new type of molecularly imprinted polymer (MIP) monolith. The imprinted monolithic column was synthesized using a mixture of carprofen (template), 4-vinylpyridine, ethylene glycol dimethacrylate, [BMIM]BF4, and chain transfer agent (CTA). Some polymerization factors, such as template-monomer molar ratio, the degree of crosslinking, the composition of the porogen, and the content of CTA, on the column efficiency and imprinting effect of the resulting MIP monolith were systematically investigated. Affinity screening of structurally similar compounds with the template can be achieved in 200 s on the MIP monolith due to high column efficiency (up to 12,070 plates/m) and good column permeability. Recognition mechanism of the imprinted monolith was also investigated.
Keywords: Carprofen; Molecularly imprinted polymers (MIP); Monolith; Ionic liquids; Reversible addition-fragmentation chain transfer

High-temperature solvent gradient interaction chromatography (HT-SGIC) is a fast and efficient fractionation technique for the chemical composition analysis of olefin copolymers. The separation of ethylene–propylene random copolymers (EPRs) was achieved on a graphitic stationary phase, Hypercarb, at 160 °C by using linear solvent gradient elution from 1-decanol to 1,2,4-trichlorobenzene (TCB). In the present work, the solvent gradient profile was modified to improve the chromatographic separation of EPRs. With the aim to obtain a better resolution in separation, a slow increase in the volume fraction of TCB was applied. This allowed for a relatively large retention region for linear polyethylene (PE) chains on the column; thereby, a broader elution volume zone between the start of the gradient and the PE elution was achieved. The efficiency of this new gradient profile was demonstrated by analysing two fully amorphous EPR samples. Clear differences in the chemical composition of these EPR samples with similar ethylene contents have been proven by using this modified solvent gradient. The comprehensive chemical composition and microstructure analysis of the SGIC-separated fractions by FTIR revealed that ethylene/propylene (EP) copolymer chains were eluted according to their ethylene/propylene contents and E or P sequence lengths, even though they are distributed in a random manner. These results showed that the solvent composition is an important factor to affect the interactive adsorption or desorption behaviour of EP chains on Hypercarb. In this way, for the first time, the determination of the complex composition and chain structure of EPR samples was achieved within short analysis time, which is not possible till now using other fractionation techniques reported. Figure A slightly modified solvent gradient method for high-temperature solvent gradient interaction chromatography (HT-SGIC) enabled the fractionation of completely amorphous ethylene–propylene rubbers (EPR) according to their microstructure with high resolution in separation. Presence of EP copolymers having short E or P blocks was identified by combing the HT-SGIC fractionation with FTIR analysis.
Keywords: Ethylene–propylene random copolymer; EP rubber; Solvent gradient interaction chromatography; Chemical composition separation

The Food and Drug Administration uses water activity behavior characteristics when adjusting test portion mass to correct for the moisture condition of its cocoa powder in-house reference material. The cocoa powder’s moisture condition, and therefore weight, equilibrates according to the relative humidity (RH) of its surroundings. This process is predictable and defined by an isotherm. The reference values in the certificate of analysis are relative to the material’s condition at 30 % RH, which is assumed to be mid-range for typical laboratory settings. Since mass variations are relatively small within a 15–50 % RH range, the mass may be measured immediately after removing a test portion from a storage bottle and used without correction if a standard uncertainty of 0.7 % is acceptable for the mass. If greater accuracy is needed and the laboratory RH is known, a very simple and quick procedure can be used whereby the test portion is left open and exposed to the laboratory air overnight before weighing. After applying a correction, the standard uncertainty for mass measurement drops to 0.3 %. Figure ᅟ
Keywords: Reference material; Water activity; Relative humidity; Isotherm

A feasibility study into the production of a freeze-dried oyster reference material for paralytic shellfish poisoning toxins by Andrew D. Turner; Adam M. Lewis; Robert G. Hatfield; Wendy A. Higman; Stephen Burrell (8621-8632).
Matrix reference materials are an essential component for the validation and quality control of analytical methodologies for the quantitation of marine biotoxins in shellfish. Given the potential advantages of reference materials in powder form, a study was conducted to assess the feasibility for the production of a freeze-dried oyster tissue reference material containing a range of important paralytic shellfish poisoning toxins. One bulk sample of a wet oyster tissue homogenate was generated following mass culturing of toxic Alexandrium and oyster feeding experiments. The bulk tissue was used to prepare untreated wet frozen aliquots with the remainder being freeze-dried and processed into appropriately-sized powder samples. A pre-column oxidation LC-FLD analysis was used to confirm the absence of any chromatographic artefacts resulting from the processing and to confirm acceptable homogeneity of the tissues. Excellent stability over both the short-term (1 month) and long-term (1 year) of the freeze-dried material was demonstrated as compared with the stability of the untreated wet tissue. A post-column oxidation LC-FLD method was used to confirm the absence of toxin epimerisation in freeze-dried tissues which were observed in the wet tissues. Overall the work showed the feasibility of an approach to produce a homogenous freeze-dried oyster matrix material with enhanced stability in comparison to the untreated wet tissue. The potential for use of the process for preparation of large scale production batches of a freeze-dried CRM for paralytic shellfish poisoning toxins has therefore been demonstrated.
Keywords: Paralytic shellfish poisoning (PSP); Oysters; Freeze drying; LC-FLD; Reference materials

Avilamycin residue in food is regulated as its marker residue dichloroisoeverninic acid (DIA). An isotope dilution liquid chromatography–tandem mass spectrometry method is established for the accurate determination of DIA in animal muscles without any pre-extraction and preconcentration prior to alkaline hydrolysis. Optimization of the sample cleanup procedures such as liquid–liquid extraction and solid phase extraction was performed by fine-tuning several critical parameters to reduce the matrix effects. Quantification of DIA in edible muscle was accomplished by using matrix-matched calibration with dichloroisoeverninic acid-d6 as internal standard. The method was validated with DIA and avilamycin-fortified poultry and porcine muscles at three different levels (25, 50, and 100 μg/kg). Conversion of avilamycin to DIA by alkaline hydrolysis was ≥92 %. The recoveries of DIA in both muscles at three fortification levels ranged from 94 to 106 % and RSDs were ≤11 % in all cases. The estimated limit of detection values in poultry and porcine muscles were 2.7 and 0.7 μg/kg, respectively. The estimated limit of quantitation values in poultry and porcine muscles were 8.3 and 2.4 μg/kg, respectively. This method is suitable for routine monitoring of avilamycin residue in food safety surveillance programs.
Keywords: Anionic mixed-mode solid phase extraction; Avilamycin; Dichloroisoeverninic acid; Isotope dilution tandem mass spectrometry

A simple, rapid, and sensitive method based on simultaneous protein precipitation and extraction of aflatoxin M1 (AFM1) followed by dispersive liquid–liquid microextraction (DLLME) and ultrahigh pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analysis was developed for the determination of AFM1 in milk samples. In order to precipitate the proteins and extract AFM1 from milk, a sample pretreatment using acetonitrile and NaCl as the extraction/denaturant solvent and salting-out agent, respectively, was optimised. Subsequently, the acetonitrile (upper) phase, containing AFM1, was used as the disperser solvent in DLLME, and extractant (chloroform) and water were added in turn to the extract to perform the DLLME process. The main parameters affecting the extraction efficiency of the whole analytical procedure, such as acetonitrile volume, amount of salt, type and volume of extractant and water volume, were carefully optimised by experimental design. Under optimum conditions, the developed method provides an enrichment factor of 33 and detection and quantification limits (0.6 and 2.0 ng kg−1, respectively) below the maximum levels imposed by current regulations for AFM1 in milk and infant milk formulae. Recoveries (61.3–75.3 %) and repeatability (RSD < 10, n = 3), tested in different types of milk at four AFM1 levels, met the performance criteria required by EC Regulation No. 401/2006. Moreover, the matrix effect on the signal intensity of the analyte was negligible. The proposed method provides a rapid extraction and an accurate determination of AFM1 in milk and formula milk using a simple and inexpensive sample preparation procedure. Figure Rapid analysis of aflatoxin M1 in milk using DLLME
Keywords: Milk; Aflatoxin M1 ; Protein precipitation; Dispersive liquid–liquid microextraction; UHPLC-MS/MS

A novel and affordable analytical setup is herein reported for automatic flow-through sorptive microextraction of organic contaminants, exploiting polydimethylsiloxane (PDMS) as a front-end to gas chromatography-ion trap-tandem mass spectrometry. The analytical procedure involves a short single-strand PDMS hollow fiber integrated in a sequential injection (SI) network for automatic fluidic handling by programmable flow. The target species are in-line extracted from 10 mL of sample containing 20 % (v/v) methanol followed by elution with a metered volume of organic solvent, which is whereupon quantitatively transferred into the programmed temperature vaporization (PTV) injector of the GC. Diffusional resistance to mass transfer was overcome by effecting the overall concentration and stripping steps at a single PDMS tubing interface. The proof of concept of the novel hyphenated system was demonstrated for extraction and determination of organochlorine pesticides (OCPs), namely, heptachlor, dieldrin, endrin, endosulfan, p,p′-dichlorodiphenyldichloroethane, p,p′-dichlorodiphenyltrichloroethane, dichlorodiphenyldichloroethylene, and endrin ketone, taken as model analytes, in environmental and industrial waters. Four organic solvents with a broad spectrum of polarity were investigated as eluents in the SI-based assembly, namely, ethyl acetate, methyl tert-butyl ether, hexane, and chloroform. Chloroform was proven the most suitable solvent for expedient elution and fast evaporation in the PTV injector. Under the selected experimental variables, limits of detection (signal-to-noise ratio (S/N) = 3) within the range of 0.3–1.1 ng L−1, limits of quantification (S/N = 10) of 1.0–3.6 ng L−1, and method repeatabilities spanning from 1.7 to 4.7 % were obtained for the suite of OCPs. The hyphenated flow analyzer was harnessed to the analysis of samples of varying matrix complexity with good relative recoveries (86–112 %) in drinking water, surface water, and influent and effluent wastewaters, with quantification limits far below those endorsed by WHO and EU drinking water directives setting maximum allowed concentrations at ≤100 ng L−1 OCPs.
Keywords: Sequential injection analysis; Polydimethylsiloxane; In-line microextraction; Gas chromatography–mass spectrometry; Organochlorine pesticides

Direct analysis in real-time mass spectrometry (DART-MS) is normally applied for small-molecule analysis up to about m/z 1,000. Here, for the analysis of polydimethylsiloxanes, high-mass capabilities expanding beyond m/z 3,000 are demonstrated. In addition, polydimethylsiloxanes provide an ideal mass calibration standard for positive-ion DART-MS. A mass reference list has been compiled to cover ions from m/z 200 up to m/z 2,600. Species with more than 20 silicon atoms exhibit increasingly broader isotopic patterns with decreasing abundances of the monoisotopic ions. The use of the first isotopic peaks for analyte ions above m/z 2,000 serves as a work-around and ensures easy and reproducible recognition of the reference peaks by the instrument data system. Here, the positive-ion DART mass spectra of polydimethylsiloxanes and the corresponding experimental procedures are described, and the mass reference list is provided.
Keywords: Direct analysis in real time (DART); Mass spectrometry; Polysiloxanes; Mass calibration; Accurate mass; Polymer analysis

Non-invasive NMR stratigraphy of a multi-layered artefact: an ancient detached mural painting by Valeria Di Tullio; Donatella Capitani; Federica Presciutti; Gennaro Gentile; Brunetto Giovanni Brunetti; Noemi Proietti (8669-8675).
NMR stratigraphy was used to investigate in situ, non-destructively and non-invasively, the stratigraphy of hydrogen-rich layers of an ancient Nubian detached mural painting. Because of the detachment procedure, a complex multi-layered artefact was obtained, where, besides layers of the original mural painting, also the materials used during the procedure all became constitutive parts of the artefact. NMR measurements in situ enabled monitoring of the state of conservation of the artefact and planning of minimum representative sampling to validate results obtained in situ by solid-state NMR analysis of the samples. This analysis enabled chemical characterization of all organic materials. Use of reference compounds and prepared specimens assisted data interpretation. Figure Right side, portable NMR scanning an ancient detached mural painting, left side NMR stratigraphy of a region of the painting
Keywords: NMR stratigraphy; NMR spectroscopy; Acrylic resins; Detached mural paintings

We describe a new use of switchable-polarity solvents for the simultaneous derivatization and extraction of triacylglycerols from vegetable oils before gas-chromatographic analysis. Different equimolecular mixtures of the commercially available amidine 1,8-diazabicyclo[5.4.0]undec-7-ene and n-alkyl alcohols were tested. Triolein was used as a model compound. Very good results were achieved by using butanol (recovery of butyl oleate was 89 ± 4 %). The procedure was applied for the characterization of the fatty acid profile of different vegetable oils. No statistically significant differences from the results obtained with the application of two traditional methods were evidenced. Moreover, the use of switchable-polarity solvents showed many advantages: owing to the basicity of the amidines, no catalyst was required; the transterification reaction was conducted under mild conditions, one step and in situ; no particular matrix interferences were evidenced; the solvent was recovered. Figure Switchable polarity solvents perform the simultaneous extraction and transesterification of triacylglycerols from vegetable oils. The method represent a new in situ derivatization procedure for the gas chromatographic characterization of thir fatty acid profiles.
Keywords: Switchable-polarity solvents; Triacylglycerols; Derivatization; Gas chromatography; Vegetable oils