Analytical and Bioanalytical Chemistry (v.405, #18)

Colin Poole (Ed.): Gas chromatography by Maria Careri (5855-5856).

Liquid chromatography–tandem mass spectrometry by Mira Petrovic; Damià Barceló (5857-5858).
is an ICREA research professor at the Catalan Institute for Water Studies (ICRA) in Girona, Spain. Her main expertise is in analytical environmental chemistry, specifically analysis of organic micropollutants by use of advanced mass spectrometric techniques and study of their fate and behaviour in the aquatic environment and during treatment of wastewater and drinking water. She has published 136 papers in SCI journals (Hirsch Index 40, 4503 citations), edited six books, and written 34 book chapters. She is included in the ISI Highly Cited in the fields of Chemistry and Environment/Ecology. is Full Research Professor at the Institute of Environmental Assessment and Water Studies IDAEA-CSIC and has been Head of the Environmental Chemistry Department (Barcelona, Spain) since 1999 and Director of the Catalan Institute of Water Research (ICRA) (Girona, Spain) since May 2008. His scientific focus is on method development and monitoring of priority, new, and emerging pollutants, in particular in water-quality assessment and management. In 2007 he was awarded the King Jaime I Prize for the Protection of Nature by the Generalitat of Valencia, Spain, for his outstanding scientific work. In 2011 he received the Prince Sultan Bin Abdulaziz International Prize for Water (PSIPW) 5th Award 2012 on Water Management and Protection, Saudi Arabia, and in 2012 the Recipharm Environmental Award, Sweden. Since 2011 he has been chairman of the Scientific and Technological Board (STB) of the European Union–Joint Programming Initiative on “Water Challenges for a Changing World”.

This paper describes the development and application of a multi-residue chiral liquid chromatography coupled with tandem mass spectrometry method for simultaneous enantiomeric profiling of 18 chiral pharmaceuticals and their active metabolites (belonging to several therapeutic classes including analgesics, psychiatric drugs, antibiotics, cardiovascular drugs and β-agonists) in surface water and wastewater. To the authors’ knowledge, this is the first time an enantiomeric method including such a high number of pharmaceuticals and their metabolites has been reported. Some of the pharmaceuticals have never been studied before in environmental matrices. Among them are timolol, betaxolol, carazolol and clenbuterol. A monitoring programme of the Guadalquivir River basin (South Spain), including 24 sampling sites and five wastewater treatment plants along the basin, revealed that enantiomeric composition of studied pharmaceuticals is dependent on compound and sampling site. Several compounds such as ibuprofen, atenolol, sotalol and metoprolol were frequently found as racemic mixtures. On the other hand, fluoxetine, propranolol and albuterol were found to be enriched with one enantiomer. Such an outcome might be of significant environmental relevance as two enantiomers of the same chiral compound might reveal different ecotoxicity. For example, propranolol was enriched with S(−)-enantiomer, which is known to be more toxic to Pimephales promelas than R(+)-propranolol. Fluoxetine was found to be enriched with S(+)-enantiomer, which is more toxic to P. promelas than R(−)-fluoxetine.
Keywords: Chiral pharmaceutical; Metabolite; LC–MS/MS; River water; Wastewater; Enantiomer

Water-soluble polar organic contaminants are discharged by rivers, cities, and ships into the oceans. Little is known on the fate, pollution effects, and thresholds of toxic chemical mixtures in the marine environment. A new trace analytical method was developed for the multi-compound analysis of polar organic chemical contaminants in marine waters. The method is based on automated solid-phase extraction (SPE) of one-liter water samples followed by ultrahigh-pressure liquid chromatography triple-quadrupole linear ion-trap mass spectrometry (UHPLC–QTRAP® MS). Marine water samples from the open Adriatic Sea taken 16 km offshore from Venice (Italy) were analyzed. Method limits of quantification (LOQs) in the low picogram per liter (pg/l) concentration range were achieved. Among the 67 target chemicals analyzed, 45 substances could be detected above the LOQ. The chemicals detected at the highest concentrations were caffeine (up to 367 ng/l), nitrophenol (36 ng/l), 2,4-dinitrophenol (34 ng/l), 5-methyl-1H-benzotriazole (18.5 ng/l), sucralose (11 ng/l), 1H-benzotriazole (9.2 ng/l), terbuthylazine (9 ng/l), alachlor (7.7 ng/l), atrazine-desisopropyl (6.6 ng/l), diethyltoluamide (DEET) (5.0 ng/l), terbuthylazine-desethyl (4.3 ng/l), metolachlor (2.8 ng/l), perfluorooctanoic acid (PFOA) (2.5 ng/l), perfluoropentanoic acid (PFPeA) (2.3 ng/l), linuron (2.3 ng/l), perfluorohexanoic acid (PFHxA) (2.2 ng/l), diuron (2.0 ng/l), perfluorohexane sulfonate (PFHxS) (1.6 ng/l), simazine (1.6 ng/l), atrazine (1.5 ng/l), and perfluorooctane sulfonate (PFOS) (1.3 ng/l). Higher concentrations were detected during summer due to increased levels of tourist activity during this period.
Keywords: Adriatic Sea; UHPLC-MS/MS; Multicompound chemical analysis; Pesticides; Pharmaceuticals; Perfluoroalkyl substances

Outcompeting GC for the detection of legacy chlorinated pesticides: online-SPE UPLC APCI/MSMS detection of endosulfans at part per trillion levels by Natalia Quinete; Jian Wang; Adolfo Fernandez; Joffre Castro; Piero R. Gardinali (5887-5899).
Endosulfan, the last remaining organochlorine pesticide, has been the subject of a number of international regulations and restriction/banning action plans worldwide. Occurrence of endosulfan residues in South Florida environments has been widely described in the literature for more than two decades. This work describes a selective, sensitive, and fast online solid-phase extraction (SPE) method coupled with liquid chromatography separation and tandem mass spectrometry (LC-MS/MS) for the determination of endosulfan isomers and endosulfan sulfate in water samples at low part per trillion levels with very little sample preparation. A negative atmospheric pressure chemical ionization source was carefully optimized to produce reproducible spectra of the target compounds with no adduct ion formation. Selected reaction monitoring transitions were monitored and quantitation was performed against a per-deuterated internal standard β-endosulfan (d4). The automated online SPE clean-up was performed using only 20 mL of untreated water sample prior to LC-MS/MS analysis. The method was capable of separating and quantifying endosulfan within a 24-min run using acetonitrile and water as mobile phases and presenting statistically calculated method detection limits of 3, 10, and 7 ng/L for endosulfan sulfate, α-endosulfan, and β-endosulfan, respectively. In addition, a QuEChERS method was successfully developed and applied for endosulfan determination in sediments/soils, floating and submerged algal mats, and small fish. Minimal matrix effects were observed in all matrices analyzed and recoveries for all analytes ranged from 50–144 %. The developed methodology was applied to monitor the occurrence and to assess the potential transport of endosulfan in the Loveland Slough watershed, an area adjacent to Everglades National Park showing long-term contamination with endosulfans.
Keywords: Endosulfan; Organochlorine insecticide; QuEChERS; Surface water; Online SPE LC-APCI-MS/MS; Fish tissue

This work introduces a liquid chromatography–electrospray ionization-hybrid quadrupole/time-of-flight mass spectrometry (LC-ESI-QTOF-MS)-based method for qualitative and quantitative analysis of poly(amidoamine) (PAMAM) dendrimers of generations 0 to 3 in an aqueous matrix. The multiple charging of PAMAM dendrimers generated by means of ESI has provided key advantages in dendrimer identification by assignation of charge state through high resolution of isotopic clusters. Isotopic distribution in function of abundance of isotopes 12C and 13C yielded valuable and complementarity data for confident characterization. A mass accuracy below 3.8 ppm for the most abundant isotopes (diagnostic ions) provided unambiguous identification of PAMAM dendrimers. Validation of the LC-ESI-QTOF-MS method and matrix effect evaluation enabled reliable and reproducible quantification. The validation parameters, limits of quantification in the range of 0.012 to 1.73 μM, depending on the generation, good linear range (R > 0.996), repeatability (RSD < 13.4 %), and reproducibility (RSD < 10.9 %) demonstrated the suitability of the method for the quantification of dendrimers in aqueous matrices (water and wastewater). The added selectivity, achieved by multicharge phenomena, represents a clear advantage in screening aqueous mixtures due to the fact that the matrix had no significant effect on ionization, with what is evidenced by an absence of sensitivity loss in most generations of PAMAM dendrimers. Fig Liquid chromatography–electrospray ionization-hybrid quadrupole/time of flight mass spectrometry (LC-ESI-QTOF-MS) based method for qualitative and quantitative analysis of PAMAM dendrimers in aqueous matrix
Keywords: PAMAM dendrimers; Aqueous matrices; Electrospray ionization; Multicharged ions; LC-ESI-QTOF-MS; Quantification

Quantitative trace analysis of fullerenes in river sediment from Spain and soils from Saudi Arabia by Josep Sanchís; Dalibor Božović; Naif A. Al-Harbi; Luis F. Silva; Marinella Farré; Damià Barceló (5915-5923).
A quantitative method based on ultrasound-assisted toluene extraction followed by liquid chromatography–electrospray ionization–tandem mass spectrometry for the analysis of C60 and C70 fullerenes, N-methylfulleropyrrolidine, [6, 6]-phenyl C61 butyric acid methyl ester and [6, 6]-thienyl C61 butyric acid methyl ester has been developed. The method was validated using fortified blank river sediments according to the criteria of Commission Decision 2002/657/EC. The method limits of detection ranged from 14 to 290 pg/g, making it suitable for its application in environmental analysis. The method has been applied to investigate fullerene content in 58 soil samples collected from different urban and industrial areas in Saudi Arabia and in river sediment from six different sites in the Llobregat River Basin. In addition, in the case of the Llobregat River, superficial water samples from the same sites of the sediments were collected and analysed using a previous method. In soils from Saudi Arabia, C60-fullerene was the only compound that was detected and quantified in 19 % of samples. In the sediments of the Llobregat River, C60-fullerene was also the only one detected (33 % of the samples), while in river water, C70-fullerene was the most frequent compound, and it was quantified in 67 % of the samples. However, C60-fullerene was present in two of the six samples, but at higher concentrations than C70-fullerene, ranging from 0.9 to 7.8 ng/L.
Keywords: Fullerene; Soils; Sediments; Liquid chromatography–mass spectrometry

An online SPE-HPLC-HESI-MS/MS method and an online SPE-HPLC-APPI-MS/MS method were developed to analyze 72 microconstituents in reclaimed water. In this study, 55 reclaimed water samples were collected from the sprinkler system for a year-long period at Florida International University Biscayne Bay Campus, where reclaimed water was reused for daily irrigation. Analysis results showed that several analytes were continuously detected in all reclaimed water samples and others will show rather transient signal increases. Coprostanol, bisphenol A, and DEET’s maximum concentration exceeded 10,000 ng/L. The four most frequently detected compounds were diphenhydramine (100 %), DEET (98 %), atenolol (98 %) and carbamazepine (96 %).
Keywords: Microcontituents; HESI; APPI; LC-MS/MS; Reclaimed water; Irrigation

In the present work, the development, optimization, and validation (including a whole stability study) of a fast, reliable, and comprehensive method for the analysis of ten anticancer drugs in hospital and urban wastewater is described. Extraction of these pharmaceutical compounds was performed using automated off-line solid-phase extraction followed by their determination by ultra-performance liquid chromatography coupled to a triple quadrupole–linear ion trap mass spectrometer. Target compounds include nine cytotoxic agents: cyclophosphamide, ifosfamide, docetaxel, paclitaxel, etoposide, vincristine, tamoxifen, methotrexate, and azathioprine; and the cytotoxic quinolone, ciprofloxacin. Method detection limits (MDL) ranged from 0.8 to 24 ng/L. Levels found of cytostatic agents in the hospital and wastewater influents did not differ significantly, and therefore, hospitals cannot be considered as the primary source of this type of contaminants. All the target compounds were detected in at least one of the influent samples analyzed: Ciprofloxacin, cyclophosphamide, tamoxifen, and azathioprine were found in most of them and achieving maximum levels of 14.725, 0.201, 0.133, and 0.188 μg/L, respectively. The rest of target cancer drugs were less frequently detected and at values ranging between MDL and 0.406 μg/L. Furthermore, a feasible, useful, and advantageous approach based on information acquisition tool (information-dependent acquisition) was used for the screening of human metabolites in hospital effluents, where the hydroxy tamoxifen, endoxifen, and carboxyphosphamide were detected.
Keywords: Cytotoxic; Anticancer drugs; Metabolites; Hospital effluent; UPLC-QqLit; IDA

An online solid-phase extraction and liquid chromatography in combination with tandem mass spectrometry method was developed for the simultaneous determination of 31 antibiotics in drinking water, surface water and reclaimed waters. The developed methodology requires small sample volume (10 mL), very little sample preparation and total sample run time was 20 min. An Ion Max API heated electrospray ionization source operated in the positive mode with two selected reaction monitoring transitions was used per antibiotic for positive identity and quantification performed by the internal standard approach, to correct for matrix effects and any losses in the online extraction step. Method detection limits were in the range of 1.2–9.7, 2.2–15, 5.5–63 ng/L in drinking water, surface water and reclaimed waters, respectively. The method accuracy in matrix spiked samples ranged from 50–150 % for the studied antibiotics. The applicability of the method was demonstrated using various environmental and reclaimed water matrices. Erythromycin was detected in more than 85 % of the samples in all matrices (28–414, n.d.–199, n.d.–66 ng/L in reclaimed, river and drinking waters respectively). The other frequently detected antibiotics in reclaimed waters were nalidixic acid, clarithromycin, azithromycin, trimethoprim, and sulfamethoxazole.
Keywords: Antibiotics; Online SPE; Reclaimed water; Surface water; Drinking water; Tandem mass spectrometry

Multi-channel PMMA microfluidic biosensor with integrated IDUAs for electrochemical detection by Nongnoot Wongkaew; Peng He; Vanessa Kurth; Werasak Surareungchai; Antje J. Baeumner (5965-5974).
A novel multi-channel poly(methyl methacrylate) (PMMA) microfluidic biosensor with interdigitated ultramicroelectrode arrays (IDUAs) for electrochemical detection was developed. The focus of the development was a simple fabrication procedure and the realization of a reliable large IDUA that can provide detection simultaneously to several microchannels. As proof of concept, five microchannels are positioned over a large single IDUA where the channels are parallel with the length of the electrode finger. The IDUAs were fabricated on the PMMA cover piece and bonded to a PMMA substrate containing the microfluidic channels using UV/ozone-assisted thermal bonding. Conditions of device fabrication were optimized realizing a rugged large IDUA within a bonded PMMA device. Gold adhesion to the PMMA, protective coatings, and pressure during bonding were optimized. Its electrochemical performance was studied using amperometric detection of potassium ferri and ferro hexacyanide. Cumulative signals within the same chip showed very good linearity over a range of 0–38 μM (R 2 = 0.98) and a limit of detection of 3.48 μM. The bonding of the device was optimized so that no cross talk between the channels was observed which otherwise would have resulted in unreliable electrochemical responses. The highly reproducible signals achieved were comparable to those obtained with separate single-channel devices. Subsequently, the multi-channel microfluidic chip was applied to a model bioanalytical detection strategy, i.e., the quantification of specific nucleic acid sequences using a sandwich approach. Here, probe-coated paramagnetic beads and probe-tagged liposomes entrapping ferri/ferro hexacyanide as the redox marker were used to bind to a single-stranded DNA sequence. Flow rates of the non-ionic detergent n-octyl-β-d-glucopyranoside for liposome lysis were optimized, and the detection of the target sequences was carried out coulometrically within 250 s and with a limit of detection of 12.5 μM. The robustness of the design and the reliability of the results obtained in comparison to previously published single-channel designs suggest that the multi-channel device offers an excellent opportunity for bioanalytical applications that require multianalyte detection and high-throughput assays. Figure Multi-channel microfluidic biosensor with integrated IDUAs for a sandwich nucleic acid hybridization assay
Keywords: Microfluidic chip; Multi-channel; Electrochemical biosensor; Ultramicroelectrode; PMMA

Enabling luminescence decay time-based sensing using integrated organic photodiodes by Martin Sagmeister; Andreas Tschepp; Elke Kraker; Tobias Abel; Bernhard Lamprecht; Torsten Mayr; Stefan Köstler (5975-5982).
The use of organic photodiodes (OPDs) for measuring phosphorescent lifetimes of optochemical oxygen sensors is described. Phosphorescent indicators with lifetimes ranging from ∼5 to 60 μs have been studied using light-emitting diodes as the excitation source and organic photodiodes integrated into the sensor substrate for detection. A measurement system using an adjusted electronic circuitry to detect photocurrents in the nanoampere range is presented. The response behaviour of the organic photodiodes has been characterized, and it was found that a forward (positive) bias had to be applied in order to decrease the response time of the OPDs to a range suitable for phosphorescence decay time measurements. A modulation cutoff frequency of ∼100 kHz has been determined, corresponding to a response time of the organic photodiodes of 1.6 μs. Two sensor dyes have been characterized regarding their lifetimes upon exposure to 0–20 % oxygen, and it was shown that results comparable to literature data and inorganic photodetectors can be achieved.
Keywords: Optical sensors; Fluorescence/luminescence; Organic photodiode; Lifetime measurement; Integrated sensor

Preparation of homogeneous samples of double-labelled protein suitable for single-molecule FRET measurements by E. Lerner; G. Hilzenrat; D. Amir; E. Tauber; Y. Garini; E. Haas (5983-5991).
Preparation of pure and homogenous site specifically single- and double-labelled biopolymers suitable for spectroscopic determination of structural characteristics is a major current challenge in biopolymers chemistry. In particular, proper analysis of single-molecule Förster resonance energy transfer measurements is based on the spectral characteristics of the probes. Heterogeneity of any of the probes may introduce errors in the analysis, and hence, care must be taken to avoid preparation of inhomogeneous labelled biopolymer samples. When we prepared samples of Escherichia coli adenylate kinase (AK) mutants labelled with either Atto 488 or Atto 647N, the products were spectrally inhomogeneous and the composition of the mixture changed gradually over time. We show here that the inhomogeneity was not a result of variation in the dye interaction with neighbouring side chains. Rather, the slow drift of the spectral characteristics of the probes was a characteristic of an irreversible chemical transformation probably due to the hydrolysis of the succinimide ring of the attached dye into its succinamic acid form. Overnight incubation of the labelled protein in mild basic solution accelerated the interconversion, yielding homogeneous labelled samples. Using this procedure, we obtained stable homogenous AK mutant labelled at residues 142 and 188.
Keywords: Single-molecule FRET; Fluorescence; Labelling; Purification

A duplex–triplex switchable DNA nanomachine was fabricated and has been applied for the demonstration of intracellular acidification and apoptosis of Ramos cells, with graphene oxide (GO) not only as transporter but also as fluorescence quencher. The machine constructed with triplex-forming oligonucleotide exhibited duplex–triplex transition at different pH conditions. By virtue of the remarkable difference in affinity of GO with single-stranded DNA and triplex DNA, and the super fluorescence quenching efficiency of GO, the nanomachine functions as a pH sensor based on fluorescence resonance energy transfer. Moreover, taking advantage of the excellent transporter property of GO, the duplex–triplex/GO nanomachine was used to sense pH changes inside Ramos cells during apoptosis. Fluorescence images showed different results between living and apoptotic cells, illustrating the potential of DNA scaffolds responsive to more complex pH triggers in living systems. Figure The caption/legend for the online abstract figure: Schematic illustration of cell apoptosis detection in Ramos cells by using duplex-triplex/GO nanocomplex
Keywords: Nucleic acid nanomachine; Cell apoptosis; Graphene oxide; Triplex; Imaging

In this study, characterization of the binding kinetics and optimization of a magnetic permeability based point-of-care (POC) immunoassay system for quantification of canine C-reactive protein (cCRP) is described. The reagent is based on a two-site heterogeneous immunoassay system utilizing conjugated superparamagnetic nanoparticles (SPION) and silica particles, both particles carrying covalently linked antibodies directed to the cCRP analyte. Detection is carried out using a magnetic permeability-based small instrument, adjusted in order to apply it in a POC setting near the patients. The kinetic parameters are characterized and applied in the final design of the assay system. In the cCRP system studied, 90 % of the binding between immobilized solid-phase silica antibody and cCRP is complete after only 15 s, and 30 s for the binding between the antibody on the SPION and the bound cCRP on the silica particle. Additionally, the binding rate constants are determined to be 149 and 30 M−1s−1, respectively. The analytical sensitivity, clinical sensitivity, and imprecision verifies the clinical usefulness of the system. Also, quantification of cCRP, using the system described, in dog clinical samples from mixed breeds shows a high correlation to a commercially available comparative cCRP ELISA system (y = 0.98 × +3.2, R 2 = 0.98, n = 47). The immunoassay system described can thus provide the veterinarian a valuable tool for rapid diagnosis and monitoring of inflammatory diseases in dogs in a setting near the patients.
Keywords: Magnetic permeability; Superparamagnetic nanoparticles; Two-site immunoassay; Canine C-reactive protein; Point-of-care

The generation of key drug metabolites for the purpose of their complete structural characterization, toxicity testing, as well as to serve as standards for quantitative studies, is a critical step in the pharmaceutical discovery and development cycle. Here, we utilized electrochemistry/mass spectrometry for the detection and subsequent generation of six phase I metabolites of simvastatin and lovastatin. Both simvastatin and lovastatin are widely used for the treatment of hypercholesterolemia. There are known drug–drug interaction issues of statin therapy, and it has been suggested that the oxidative metabolites may contribute to the cholesterol-lowering effect of both statins. Of the known phase I metabolites of simvastatin and lovastatin, none are commercially available, and chemical means for the synthesis of a very few of them have been previously reported. Here, we report that electrochemical oxidation of less than 1 mg each of simvastatin and lovastatin led to the generation of three oxidative metabolites of each parent to allow complete nuclear magnetic resonance characterization of all six metabolites. The yields obtained by the electrochemical approach were also compared with incubation of parent drug with commercially available bacterial mutant CYP102A1 enzymes, and it was found that the electrochemical approach gave higher yields than the enzymatic oxidations for the generation of most of the observed oxidative metabolites in this study. Figure Generation of statin drug metabolites by EC/MS (representative mass voltammogram shown), and recombinant CYP enzymes
Keywords: Electrochemistry; Electrochemistry/mass spectrometry (EC/MS); Metabolite generation; Simvastatin; Lovastatin; CYP102A1

11-Nor-9-carboxy-∆9-tetrahydrocannabinol quantification in human oral fluid by liquid chromatography–tandem mass spectrometry by Karl B. Scheidweiler; Sarah K. Himes; Xiaohong Chen; Hua-Fen Liu; Marilyn A. Huestis (6019-6027).
Currently, ∆9-tetrahydrocannabinol (THC) is the analyte quantified for oral fluid cannabinoid monitoring. The potential for false-positive oral fluid cannabinoid results from passive exposure to THC-laden cannabis smoke raises concerns for this promising new monitoring technology. Oral fluid 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THCCOOH) is proposed as a marker of cannabis intake since it is not present in cannabis smoke and was not measureable in oral fluid collected from subjects passively exposed to cannabis. THCCOOH concentrations are in the picogram per milliliter range in oral fluid and pose considerable analytical challenges. A liquid chromatography–tandem mass spectrometry (LCMSMS) method was developed and validated for quantifying THCCOOH in 1 mL Quantisal-collected oral fluid. After solid phase extraction, chromatography was performed on a Kinetex C18 column with a gradient of 0.01 % acetic acid in water and 0.01 % acetic acid in methanol with a 0.5-mL/min flow rate. THCCOOH was monitored in negative mode electrospray ionization and multiple reaction monitoring mass spectrometry. The THCCOOH linear range was 12–1,020 pg/mL (R 2 > 0.995). Mean extraction efficiencies and matrix effects evaluated at low and high quality control (QC) concentrations were 40.8–65.1 and −2.4–11.5 %, respectively (n = 10). Analytical recoveries (bias) and total imprecision at low, mid, and high QCs were 85.0–113.3 and 6.6–8.4 % coefficient of variation, respectively (n = 20). This is the first oral fluid THCCOOH LCMSMS triple quadrupole method not requiring derivatization to achieve a <15 pg/mL limit of quantification. The assay is applicable for the workplace, driving under the influence of drugs, drug treatment, and pain management testing. Figure Multiple reaction monitoring ion chromatograms for THCCOOH quantifier and qualifier transitions: from a blank oral fluid sample (A and B), blank oral fluid fortified at the 12 pg/mL limit of quantification (C and D) and an authentic specimen containing 26 pg/mL THCCOOH collected 0.25 h after 5 mg oral Marinol (E andF)
Keywords: Cannabinoids; Carboxy THC; Oral fluid; Metabolites; Analytical method; LCMSMS

Several in vitro methods have been tested for their ability to predict drug penetration across the blood–brain barrier (BBB) into the central nervous system (CNS). In this article, the performance of a variety of micellar liquid chromatographic (MLC) methods and immobilized artificial membrane (IAM) liquid chromatographic approaches were compared for a set of 45 solutes. MLC measurements were performed on a C18 column with sodium dodecyl sulfate (SDS), polyoxyethylene (23) lauryl ether (Brij35), or sodium deoxycholate (SDC) as surfactant in the micellar mobile phase. IAM liquid chromatography measurements were performed with Dulbecco’s phosphate-buffered saline (DPBS) and methanol as organic modifier in the mobile phase. The corresponding retention and computed descriptor data for each solute were used for construction of models to predict transport across the blood–brain barrier (log BB). All data were correlated with experimental log BB values and the relative performance of the models was studied. SDS-based models proved most suitable for prediction of log BB values, followed closely by a simplified IAM method, in which it could be observed that extrapolation of retention data to 0 % modifier in the mobile phase was unnecessary.
Keywords: Blood–brain barrier; Central nervous system; Micellar liquid chromatography; Immobilized artificial membrane; Modeling

It is well known that enoxaparin, a widely used anticoagulant and low-molecular-weight heparin containing a large number of oligosaccharides, possesses anti-inflammatory activity. Whilst enoxaparin has shown promising results in various inflammatory disorders, some of its oligosaccharides have anti-inflammatory properties and others increase the risk of bleeding due to their anticoagulant effects. The aim of this study was to develop an effective ion exchange chromatographic (IC) technique which allows the separation, isolation and, consequently, the identification of different oligosaccharides of enoxaparin with or without anticoagulant activity. The developed method utilises a semi-preparative CarboPac PA100 (9 × 250 mm) ion exchange column with sodium chloride gradient elution and UV detection at 232 nm. The method successfully resolved enoxaparin into more than 30 different peaks. IC-derived oligosaccharides with high, moderate, low or no anticoagulant activity were identified using an anti-factor Xa assay. The anti-inflammatory activity of selected oligosaccharides was investigated using the Griess assay. Using this technique, the oligosaccharides of enoxaparin with low or no anticoagulant activity, whilst exhibiting significant anti-inflammatory activity, could be fractionated. This technique can provide a platform to identify the oligosaccharides which are devoid of significant anticoagulant activity and are responsible for the therapeutic effects of enoxaparin that have been observed in various inflammatory conditions. Figure Determination of approximate saccharide composition of ion exchange chromatography separated enoxaparin
Keywords: Ion exchange chromatography; Enoxaparin; Anticoagulant oligosaccharides; Non-anticoagulant oligosaccharides; Anti-inflammatory activity

Capillary electrophoretic analysis of hydroxyl radicals produced by respiring mitochondria by Margaret A. Donoghue; Xin Xu; David A. Bernlohr; Edgar A. Arriaga (6053-6060).
Here, we report the use of a capillary electrophoretic method with laser-induced fluorescence detection to evaluate hydroxyl radicals produced by respiring mitochondria. The probe, hydroxyphenylfluorescein (HPF), is separated from the product, fluorescein, in under 5 min with zeptomole and attomole limits of detection for fluorescein and HPF, respectively. Purification of the probe with a C-18 SPE column is necessary to reduce the fluorescein impurity in the probe stock solution from 0.4 % to less than 0.001 %. HPF was responsive to hydroxyl radicals produced by isolated mitochondria from L6 cells, and this signal was blunted when DMSO was added to scavenge hydroxyl radicals and when carbonyl cyanide m-chlorophenylhydrazone was added to depolarize the mitochondria. The method was used to compare hydroxyl radical levels in mitochondria isolated from brown adipose tissue of lean and obese mice. Mitochondria from obese mice produced significantly more hydroxyl radicals than those from lean mice. Figure Caption for figure abstract: Mitochondria are the main source of cellular reactive oxygen species. While all are of interest, the specific detection of hydroxyl radicals can be achieved with the fluorescent probe HPF. MEKC-LIF is used to separate the probe HPF from its product, fluorescein. This was demonstrated by treating enriched mitochondria fractions from L6 cells with HPF. Electropherograms show an increase in fluorescein peak area when mitochondria are stimulated with 100 μM Fe2+.
Keywords: Hydroxyl radicals; Mitochondria; Obesity; Micellar electrokinetic chromatography; Capillary electrophoresis

Hard X-ray nanoprobe investigations of the subtissue metal distributions within Daphnia magna by B. De Samber; K. A. C. De Schamphelaere; C. R. Janssen; B. Vekemans; R. De Rycke; G. Martinez-Criado; R. Tucoulou; P. Cloetens; L. Vincze (6061-6068).
The unique potential of nanoscale elemental imaging of major/minor and trace-level elemental distributions within thin biological tissue sections of the ecotoxicological model organism Daphnia magna is demonstrated by synchrotron radiation nano-X-ray fluorescence (nano-XRF). The applied highly specialized sample preparation method, coupled with the high spatial resolution (∼180 nm) and high X-ray photon flux (6 × 1011 photons/s) available at the European Synchrotron Radiation Facility (ESRF) ID22NI beamline proved to be critical for the high-quality visualization of (trace-)metal distributions on the submicron level within the target structures of interest. These include the branchial sacs on the thoracic appendages (epipodites) of D. magna, which are osmoregulatory regions where ion exchange occurs. For the main element of interest (Zn), detection limits of 0.7 ppm (3 ag) was reached in fast-scanning mode using an acquisition time of 0.3 s/pixel. As demonstrated, synchrotron radiation nano-XRF revealed the elemental distributions of Ca, Fe, and Zn within this osmoregulatory region on the submicron scale, aiding the exploration of possible detoxification mechanisms of Zn within D. magna at the subtissue level.
Keywords: Nano-XRF; Ecotoxicology; Daphnia magna ; Metallomics; OsO4 staining

High mass accuracy and high mass resolving power FT-ICR secondary ion mass spectrometry for biological tissue imaging by Donald F. Smith; Andras Kiss; Franklin E. Leach III; Errol W. Robinson; Ljiljana Paša-Tolić; Ron M. A. Heeren (6069-6076).
Biological tissue imaging by secondary ion mass spectrometry has seen rapid development with the commercial availability of polyatomic primary ion sources. Endogenous lipids and other small bio-molecules can now be routinely mapped on the sub-micrometer scale. Such experiments are typically performed on time-of-flight mass spectrometers for high sensitivity and high repetition rate imaging. However, such mass analyzers lack the mass resolving power to ensure separation of isobaric ions and the mass accuracy for elemental formula assignment based on exact mass measurement. We have recently reported a secondary ion mass spectrometer with the combination of a C60 primary ion gun with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) for high mass resolving power, high mass measurement accuracy, and tandem mass spectrometry capabilities. In this work, high specificity and high sensitivity secondary ion FT-ICR MS was applied to chemical imaging of biological tissue. An entire rat brain tissue was measured with 150 μm spatial resolution (75 μm primary ion spot size) with mass resolving power (mm 50%) of 67,500 (at m/z 750) and root-mean-square measurement accuracy less than two parts-per-million for intact phospholipids, small molecules and fragments. For the first time, ultra-high mass resolving power SIMS has been demonstrated, with mm 50% > 3,000,000. Higher spatial resolution capabilities of the platform were tested at a spatial resolution of 20 μm. The results represent order of magnitude improvements in mass resolving power and mass measurement accuracy for SIMS imaging and the promise of the platform for ultra-high mass resolving power and high spatial resolution imaging. Figure C60 secondary ion FT-ICR MS provides unprecedented mass resolving power and mass accuracy for SIMS imaging of biological tissue sections. Overlaid selected ion images from rat brain (left) and high spatial resolution imaging of organic dye underneath a TEM grid (right).
Keywords: Imaging mass spectrometry; High resolution mass spectrometry; SIMS; C60 ; Accurate mass

Acebutolol and alprenolol metabolism predictions: comparative study of electrochemical and cytochrome P450-catalyzed reactions using liquid chromatography coupled to high-resolution mass spectrometry by Ugo Bussy; Marcel Delaforge; Chaimaâ El-Bekkali; Véronique Ferchaud-Roucher; Michel Krempf; Illa Tea; Nicolas Galland; Denis Jacquemin; Mohammed Boujtita (6077-6085).
A comparative study of the electrochemical conversion and the biotransformation performed by the cytochrome P450 (CYP450) obtained by rat liver microsomes has been achieved to elucidate the oxidation mechanism of both acebutolol and alprenolol. For this purpose, a wide range of reactions such as N-dealkylation, O-dealkoxylation, aromatic hydroxylation, benzyl hydroxylation, alkyl hydroxylation, and aromatic hydroxylation have been examined in this study, and their mechanisms have been compared. Most of the results of the electrochemical oxidation have been found to be in accordance with those obtained by incubating acebutolol and alprenolol in the presence of CYP450, i.e., N-dealkylation, benzyl hydroxylation, and O-dealkoxylation reactions catalyzed by liver microsomes were found to be predicted by the electrochemical oxidation. The difficulty for the electrochemical process to mimic both aromatic and alkyl hydroxylation reactions has also been discussed, and the hypothesis for the absence of aromatic hydroxylated and alkyl hydroxylated products, respectively, for alprenolol and acebutolol, under the anodic oxidation has been supported by theoretical calculation. The present study highlights the potential and limitation of coupling of electrochemistry–liquid chromatography–high-resolution mass spectrometry for the study of phase I and phase II reactions of acebutolol and alprenolol. Figure The electrochemical conversion versus the biotransformation catalyzed by CYP450
Keywords: EC-MS; EC-LC-HRMS; CYP450; Drug metabolism prediction; β-Blockers

Comparison of GC stationary phases for the separation of fatty acid methyl esters in biodiesel fuels by Julian C. Goding; Dorisanne Y. Ragon; Jack B. O’Connor; Sarah J. Boehm; Amber M. Hupp (6087-6094).
The fatty acid methyl ester (FAME) content of biodiesel fuels has traditionally been determined using gas chromatography with a polar stationary phase. In this study, a direct comparison of the separation of FAMEs present in various biodiesel samples on three polar stationary phases and one moderately polar stationary phase (with comparable column dimensions) was performed. Retention on each column was based on solubility in and polarity of the phase. Quantitative metrics describing the resolution of important FAME pairs indicate high resolution on all polar columns, yet the best resolution, particularly of geometric isomers, is achieved on the cyanopropyl column. In addition, the separation of four C18 monounsaturated isomers was optimized and the elution order determined on each column. FAME composition of various biodiesel fuel types was determined on each column to illustrate (1) chemical differences in biodiesels produced from different feedstocks and (2) chemical similarities in biodiesels of the same feedstock type produced in different locations and harvest seasons.
Keywords: GC-MS; Fatty acid methyl esters; Biodiesel fuels; Cyanopropyl phase; Polyethyelene glycol phase; 35 % phenyl phase

Application of normal-phase high-performance liquid chromatography followed by gas chromatography for analytics of diesel fuel additives by Grzegorz Boczkaj; Mariusz Jaszczołt; Andrzej Przyjazny; Marian Kamiński (6095-6103).
The paper presents the results of investigations on new procedures of determination of selected cleaning additives in diesel fuel. Two procedures: one-step analysis using gas chromatography with flame ionization detection (GC-FID) or mass spectrometry (GC-MS) and a two-step procedure in which normal-phase high-performance liquid chromatography (NP-HPLC) was used for preliminary separation of the additives, were compared. The additive fraction was collected using either simple elution or eluent backflush. Final determinations were performed by GC-FID and GC-MS. The studies revealed that it was impossible to determine the investigated analytes by one-step procedures, i.e. by using solely HPLC or GC. On the other hand, the use of a two-step procedure ensures reproducible results of determinations, and the limits of quantitation are, depending on the method of fraction collection by HPLC, from 1.4–2.2 ppm (GC-MS in SIM mode) to 9.6–24.0 ppm (GC-FID). Precision and accuracy of the developed procedures are compared, and possible determination errors and shortcomings discussed. Figure Overview of the developed method
Keywords: Fuels; HPLC; GC; Mass spectrometry; Diesel; Sample preparation; Additives

A simple, rapid, and environmentally friendly HPLC method was developed and validated for the separation of four compounds (4-aminophenol, caffeine, paracetamol, and propyphenazone) with different chemical properties. A “green” mobile phase, employing water as the major eluent, was proposed and applied to the separation of analytes with different polarity on polyethylene glycol (PEG) stationary phase. The chromatography separation of all compounds and internal standard benzoic acid was performed using isocratic elution with a low-toxicity mobile phase consisting of 0.04 % (v/v) triethylamine and water. HPLC separation was carried out using a PEG reversed-phase stationary phase Supelco Discovery HS PEG column (15 × 4 mm; particle size 3 μm) at a temperature of 30 °C and flow rate at 1.0 mL min−1. The UV detector was set at 210 nm. In this study, a PEG stationary phase was shown to be suitable for the efficient isocratic separation of compounds that differ widely in hydrophobicity and acid–base properties, particularly 4-aminophenol (log P, 0.30), caffeine (log P, −0.25), and propyphenazone (log P, 2.27). A polar PEG stationary phase provided specific selectivity which allowed traditional chromatographic problems related to the separation of analytes with different polarities to be solved. The retention properties of the group of structurally similar substances (aromatic amines, phenolic compounds, and xanthine derivatives) were tested with different mobile phases. The proposed green chromatography method was successfully applied to the analysis of active substances and one degradation impurity (4-aminophenol) in commercial preparation. Under the optimum chromatographic conditions, standard calibration was carried out with good linearity correlation coefficients for all compounds in the range (0.99914–0.99997, n = 6) between the peak areas and concentration of compounds. Recovery of the sample preparation was in the range 100 ± 5 % for all compounds. The intraday method precision was determined as RSD, and the values were lower than 1.00 %. Green chromatography approach – advantages of the technique
Keywords: Green chromatography; Polyethylene glycol stationary phase (PEG); 4-Aminophenol; Caffeine; Paracetamol; Propyphenazone

In this study, levels of esterified and nonesterified fatty acids (EFAs and NEFAs, respectively) were compared in obese individuals (body mass index between 30 and 47 kg m–2) in basal state and after intake of four different breakfasts prepared with oils heated at frying temperature. The target oils were three sunflower oils—pure, enriched with dimethylsiloxane (400 μg mL–1) as lipophilic oxidation inhibitor, and enriched with phenolic compounds (400 μg mL–1) as hydrophilic oxidation inhibitors—and virgin olive oil with a natural content of phenolic compounds of 400 μg mL–1. The intake of breakfasts was randomized to avoid trends associated to this variability source. EFAs and NEFAs were subjected to a sequential derivatization step for independent gas chromatography–mass spectrometry analysis of both fractions of metabolites in human serum. Derivatization was assisted by ultrasonic energy to accelerate the reaction kinetics, as required for high-throughput analysis. Statistical analysis supported on univariate (multifactor ANOVA) and multivariate approaches (principal component analysis and partial least squares–discriminant analysis) allowed identification of the main variability sources and also discriminating between individuals after intake of each breakfast. Individuals’ samples after intake of breakfasts prepared with virgin olive oil were clearly separated from those who ingested the remaining breakfasts. The main compounds contributing to discrimination were omega-3 and omega-6 EFAs with special emphasis on arachidonic acid and eicosapentaenoic acid. These two polyunsaturated fatty acids are the precursors of eicosanoid metabolites, which are of vital importance as they play important roles in inflammation and in the pathogenesis of vascular and malignant diseases as cancer.
Keywords: Deep frying; Dietary intervention; Serum; Esterified fatty acids; Nonesterified fatty acids; Targeted nutrimetabolomics; Principal components analysis

SERS-based DNA detection in aqueous solutions using oligonucleotide-modified Ag nanoprisms and gold nanoparticles by Min Liu; Zhuyuan Wang; Shenfei Zong; Ruohu Zhang; Dan Zhu; Shuhong Xu; Chunlei Wang; Yiping Cui (6131-6136).
Oligonucleotide-modified nanoparticle conjugates show highly promising potential for SERS-based DNA detection. However, it remains challenging to carry out the SERS-based DNA detection in aqueous solutions directly using oligonucleotide-modified nanoparticles, because the Raman reporters would exhibit lower signals when they are dispersed in aqueous solutions than laid on “dry” metal nanoparticles. Here, we synthesized stable oligonucleotide-modified Ag nanoprism conjugates, and performed SERS-based DNA detection in aqueous solution directly by using such conjugates in combination with Raman reporter-labeled, oligonucleotide-modified gold nanoparticles. The experimental results indicate that this SERS-based DNA detection approach exhibited a good linear correlation between SERS signal intensity and the logarithm of target DNA concentration ranging from 10−11∼10−8 M. This sensitivity is comparable to those SERS-based DNA detection approaches with the “dry” process. Additionally, a similar correlation could also be observed in duplex target DNA detection by SERS hybrid probes. Our results suggest that the oligonucleotide-modified Ag nanoprisms may be developed as a powerful SERS-based DNA detection tool. Scheme of SERS-based DNA detection in aqueous solutions. Capture DNA-modified Ag nanoprisms and Raman reporter-labeled, report DNA-modified gold nanoparticles are utilized in the detection
Keywords: DNA; Silver nanoprism; Oligonucleotide; SERS; Gold nanoparticle

Validation of a novel derivatization method for GC–ECD determination of acrylamide in food by Ivan Notardonato; Pasquale Avino; Angela Centola; Giuseppe Cinelli; Mario Vincenzo Russo (6137-6141).
This paper proposes a new method for quantitative analysis of acrylamide in cereal-based foods and potato chips. The method uses reaction with trifluoroacetic anhydride, and analyses the resulting derivative by use of gas chromatography with electron-capture detection (GC–ECD). The effects of derivatization conditions, including temperature, reaction time, and catalyst, on the acylation reaction were evaluated. Chromatographic analysis was performed on an SE-54 capillary column. Under the optimum conditions, good retention and peak response were achieved for the acrylamide derivative. The analytical method was fully validated by assessment of LODs and LOQs (1 ng g−1 and 25 ng g−1, with relative standard deviations (RSD) 2.1 and 3.6, respectively), linearity (R = 0.9935 over the range 0.03–10 μg g−1), and extraction recovery (>96 %, with RSD below 2.0, for acrylamide spiked at 1, 20, 50, and 100 ng g−1; 99.8 % for acrylamide content >1000 ng g−1). The method requires no clean-up of the acrylamide derivative before injection. The method has been successfully used to determine acrylamide levels in different commercial cereal-based foods, French fries, and potato chips. Figure Novel derivatization method and GC-ECD analysis of acrylamide in cooked foods
Keywords: Acrylamide; Derivatization reaction; Trifluoroacetic anhydride; Cereal-based food; GC–ECD