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Analytical and Bioanalytical Chemistry (v.405, #10)

Keith R. Jennings (Ed.): A history of European mass spectrometry by Witold Danikiewicz (pp. 3011-3012).

Microchip-based electrochemical detection for monitoring cellular systems by Alicia S. Johnson; Asmira Selimovic; R. Scott Martin (pp. 3013-3020).

The use of microchip devices to study cellular systems is a rapidly growing research area. There are numerous advantages of using on-chip integrated electrodes to monitor various cellular processes. The purpose of this review is to give examples of advancements in microchip-based cellular analysis, specifically where electrochemistry is used for the detection scheme. These examples include on-chip detection of single-cell quantal exocytosis, electrochemical analysis of intracellular contents, the ability to integrate cell culture/immobilization with electrochemistry, and the use of integrated electrodes to ensure cell confluency in longer-term cell culture experiments. A perspective on future trends in this area is also given.

Keywords: Microchip; Microelectrode; Cell culture; Single-cell analysis

Applications of monolithic solid-phase extraction in chromatography-based clinical chemistry assays by Dustin R. Bunch; Sihe Wang (pp. 3021-3033).

Complex matrices, for example urine, serum, plasma, and whole blood, which are common in clinical chemistry testing, contain many non-analyte compounds that can interfere with either detection or in-source ionization in chromatography-based assays. To overcome this problem, analytes are extracted by protein precipitation, solid-phase extraction (SPE), and liquid–liquid extraction. With correct chemistry and well controlled material SPE may furnish clean specimens with consistent performance. Traditionally, SPE has been performed with particle-based adsorbents, but monolithic SPE is attracting increasing interest of clinical laboratories. Monoliths, solid pieces of stationary phase, have bimodal structures consisting of macropores, which enable passage of solvent, and mesopores, in which analytes are separated. This structure results in low back-pressure with separation capabilities similar to those of particle-based adsorbents. Monoliths also enable increased sample throughput, reduced solvent use, varied support formats, and/or automation. However, many of these monoliths are not commercially available. In this review, application of monoliths to purification of samples from humans before chromatography-based assays will be critically reviewed.

Keywords: Clinical chemistry; Monolith; Solid-phase extraction

Advances in glycosaminoglycanomics by ¹⁵N-NMR spectroscopy by Vitor H. Pomin (pp. 3035-3048).

Recent developments to enhance sensitivity in solution NMR spectroscopy such as the advent and spread of the use of high magnetic fields, cryoprobe technology, isotopic labeling techniques, and new combinations of 2D experiments have pushed the limits in structural NMR analysis of glycosaminoglycans (GAGs). This review is dedicated to the less sensitive ¹⁵N isotope of hexosamines rather than the commonly used anomeric and ring ¹H and ¹³C resonances of uronic acids and hexosamines. Given that GAG types are basically classified on the basis of their composing hexosamine types together with variations of their sulfation patterns, and epimerized forms of the adjacent uronic acids, ¹⁵N-related NMR studies on native GAGs, oligosaccharides, or the various composing amino sugars have proved to be quite useful in the retrieval of both structural and dynamic information, despite the low number of resultant peaks. This in turn reduces significantly chemical shift degeneracy and at the same time facilitates spin and structural assignments. This review covers the principal contributions made so far by solution ¹⁵N-NMR spectroscopy to progress in the structural biology of GAGs in the current glycomics age. Figure Glycosaminoglycans and the typical position of their ¹H-¹⁵N resonances

Keywords: Amino sugars; Biomolecular NMR; Glycosaminoglycans; ¹⁵N-NMR

Determination of sulfite with emphasis on biosensing methods: a review by Chandra Shekhar Pundir; Rachna Rawal (pp. 3049-3062).

Sulfite is used as a preservative in a variety of food and pharmaceutical industries to inhibit enzymatic and nonenzymatic browning and in brewing industries as an antibacterial and antioxidizing agent. Convenient and reproducible analytical methods employing sulfite oxidase are an attractive alternative to conventional detection methods. Sulfite biosensors are based on measurement of either O₂ or electrons generated from splitting of H₂O₂ or heat released during oxidation of sulfite by immobilized sulfite oxidase. Sulfite biosensors can be grouped into 12 classes. They work optimally within 2 to 900 s, between pH 6.5 and 9.0, 25 and 40 °C, and in the range from 0 to 50,000 μM, with detection limit between 0.2 and 200 μM. Sulfite biosensors measure sulfite in food, beverages, and water and can be reused 100–300 times over a period of 1–240 days. The review presents the principles, merits, and demerits of various analytical methods for determination of sulfite, with special emphasis on sulfite biosensors.

Keywords: Sulfite; Sulfite biosensors; Sulfite oxidase; Food; Beverages and water

Honey protein extraction and determination by mass spectrometry by Lee Suan Chua; Jun You Lee; Giek Far Chan (pp. 3063-3074).

There are relatively limited studies on the protein of honey samples mainly because of the low amount of protein in honey (0.1–0.5 %), the difficulty in extracting honey protein from the sugar-rich environment, and the hindrance of protein characterization by conventional approaches. Several protein extraction methods such as mechanical (ultrafiltration and ultracentrifugation) and chemical (precipitation) techniques have been applied to different types of honey samples. Most of these studies reported the quantity and molecular size of honey protein from gel electrophoresis, but were unable to identify and characterize the protein. This limitation might be due to the low capacity of analytical equipment in those days. Although different precipitants have also been used, not all them are compatible with mass spectrometric methods during downstream analysis. As a result, the sample preparation step is essential in order to confidently characterize the low and varied amount of honey protein. Nowadays, honey protein is getting attention from researchers because of its potential activity in pharmacological applications. Therefore, honey protein extraction and determination by mass spectrometry are critically reviewed in order to stimulate further honey protein research. Figure Flow of protein extraction and identification from honey samples

Keywords: Honey protein; Protein extraction; Mass spectrometry; Database search

Prevention of a dangerous tendency in the presentation of the results of GC-MS identification by Igor G. Zenkevich (pp. 3075-3083).

is head of the Laboratory of Gas Chromatography at the chair of Organic Chemistry (St. Petersburg State University, Department of Chemistry, St. Petersburg, Russian Federation). Besides teaching the students, the principal area of his scientific interests includes different aspects of chromatographic and chromato-spectral identification of organic compounds using combinations of analytical parameters, theory of identification, precalculation the values of physicochemical properties, chemometrics, and related problems. Since 1999 he has collaborated with National Institute of Standards & Technology (NIST, USA) in the establishing the GC retention indices database.

Simultaneous chromatography and electrophoresis: two-dimensional planar separations by Peter R. Stevenson; Bret E. Dunlap; Paul S. Powell; Brae V. Petersen; Christopher J. Hatch; Hung Chan; Garret I. Still; Michael T. Fulton; Justin S. McKell; David C. Collins (pp. 3085-3089).

Single-dimension separations are routinely coupled in series to achieve two-dimensional separations, yet little has been done to simultaneously exploit multiple dimensions during separation. In this work, simultaneous chromatography and electrophoresis is introduced and evaluated for its potential to achieve two-dimensional separations. In simultaneous chromatography and electrophoresis, chromatography occurs via capillary action while an orthogonal electric field concurrently promotes electrophoresis in a second dimension. A novel apparatus with a dual solvent reservoir was designed to apply the concurrent electric field. Various compounds were used to characterize the apparatus and technique, i.e., vitamins, amino acids, and dyes. Improved separation is reported with equivalent analysis times in comparison to planar chromatography alone. The feasibility of simultaneously employing chromatography and electrophoresis in two dimensions is discussed. Figure Separation of eight dyes is improved in comparison to (a) planar chromatography alone when employing (b) simultaneous chromatography and electrophoresis

Keywords: Planar chromatography; Amino acids; Separations/instrumentation; Electrophoresis

Gold internal standard correction for elemental imaging of soft tissue sections by LA-ICP-MS: element distribution in eye microstructures by Ioana Konz; Beatriz Fernández; M. Luisa Fernández; Rosario Pereiro; Héctor González; Lydia Álvarez; Miguel Coca-Prados; Alfredo Sanz-Medel (pp. 3091-3096).

Laser ablation coupled to inductively coupled plasma mass spectrometry has been developed for the elemental imaging of Mg, Fe and Cu distribution in histological tissue sections of fixed eyes, embedded in paraffin, from human donors (cadavers). This work presents the development of a novel internal standard correction methodology based on the deposition of a homogeneous thin gold film on the tissue surface and the use of the ¹⁹⁷Au⁺ signal as internal standard. Sample preparation (tissue section thickness) and laser conditions were carefully optimized, and internal normalisation using ¹⁹⁷Au⁺ was compared with ¹³C⁺ correction for imaging applications. ²⁴Mg⁺, ⁵⁶Fe⁺ and ⁶³Cu⁺ distributions were investigated in histological sections of the anterior segment of the eye (including the iris, ciliary body, cornea and trabecular meshwork) and were shown to be heterogeneously distributed along those tissue structures. Reproducibility was assessed by imaging different human eye sections from the same donor and from ten different eyes from adult normal donors, which showed that similar spatial maps were obtained and therefore demonstrate the analytical potential of using ¹⁹⁷Au⁺ as internal standard. The proposed analytical approach could offer a robust tool with great practical interest for clinical studies, e.g. to investigate trace element distribution of metals and their alterations in ocular diseases. Figure Development of a new internal standard correction methodology for qualitative elemental imaging by LA-ICP-MS

Keywords: Laser ablation; Biological samples; Mass spectrometry/ICP-MS

Electrochemical genosensor for detection of human mammaglobin in polymerase chain reaction amplification products of breast cancer patients by Xiongwei Xu; Xiuhua Weng; Ailin Liu; Qihuang Lin; Changlian Wang; Wei Chen; Xinhua Lin (pp. 3097-3103).

Human mammaglobin (MG) has been found to be the most specific molecular marker for the hematogenous spread of breast cancer cells. In our study, an electrochemical impedance spectroscopic DNA biosensor was established for the detection of MG in breast cancer patients. The working conditions for the biosensor, such as immobilization time, rinse process, and hybridization process, were optimized. Under the optimal conditions, the charge transfer resistance of the proposed DNA biosensor shows excellent correlation with the amount of the complementary oligonucleotides in the range from 1.0 × 10⁻⁹ to 2.0 × 10⁻⁸ M. The detection limit is 5.0 × 10⁻¹⁰ M. The proposed biosensor was used to detect the polymerase chain reaction amplification products of actual clinical breast cancer samples. The results were compared with that obtained by conventional gel electrophoresis. The results indicate that the electrochemical impedance spectroscopic assay is significantly sensitive and time-saving. The simple strategy described here is expected to be used in clinical application for early diagnosis of breast cancer. Figure The specificity of the electrochemical biosensor for the PCR products investigated by comparing the detection results of positive sample and negative control.

Keywords: Electrochemical impedance spectroscopy; Polymerase chain reaction; Biosensor; Human mammaglobin; Breast cancer

Evaluation of needle trap micro-extraction and automatic alveolar sampling for point-of-care breath analysis by Phillip Trefz; Lisa Rösner; Dietmar Hein; Jochen K. Schubert; Wolfram Miekisch (pp. 3105-3115).

Needle trap devices (NTDs) have shown many advantages such as improved detection limits, reduced sampling time and volume, improved stability, and reproducibility if compared with other techniques used in breath analysis such as solid-phase extraction and solid-phase micro-extraction. Effects of sampling flow (2–30 ml/min) and volume (10–100 ml) were investigated in dry gas standards containing hydrocarbons, aldehydes, and aromatic compounds and in humid breath samples. NTDs contained (single-bed) polymer packing and (triple-bed) combinations of divinylbenzene/Carbopack X/Carboxen 1000. Substances were desorbed from the NTDs by means of thermal expansion and analyzed by gas chromatography-mass spectrometry. An automated CO₂-controlled sampling device for direct alveolar sampling at the point-of-care was developed and tested in pilot experiments. Adsorption efficiency for small volatile organic compounds decreased and breakthrough increased when sampling was done with polymer needles from a water-saturated matrix (breath) instead from dry gas. Humidity did not affect analysis with triple-bed NTDs. These NTDs showed only small dependencies on sampling flow and low breakthrough from 1–5 %. The new sampling device was able to control crucial parameters such as sampling flow and volume. With triple-bed NTDs, substance amounts increased linearly with increasing sample volume when alveolar breath was pre-concentrated automatically. When compared with manual sampling, automatic sampling showed comparable or better results. Thorough control of sampling and adequate choice of adsorption material is mandatory for application of needle trap micro-extraction in vivo. The new CO₂-controlled sampling device allows direct alveolar sampling at the point-of-care without the need of any additional sampling, storage, or pre-concentration steps.

Keywords: Sampling; Clinical/biomedical analysis; Breath analysis; Air/gases; Needle-trap-micro-extraction; GC-MS

Cloud point extraction of Δ⁹-tetrahydrocannabinol from cannabis resin by S. Ameur; B. Haddou; Z. Derriche; J. P. Canselier; C. Gourdon (pp. 3117-3123).

A cloud point extraction coupled with high performance liquid chromatography (HPLC/UV) method was developed for the determination of Δ⁹-tetrahydrocannabinol (THC) in micellar phase. The nonionic surfactant “Dowfax 20B102” was used to extract and pre-concentrate THC from cannabis resin, prior to its determination with a HPLC–UV system (diode array detector) with isocratic elution. The parameters and variables affecting the extraction were investigated. Under optimum conditions (1 wt.% Dowfax 20B102, 1 wt.% Na₂SO₄, T = 318 K, t = 30 min), this method yielded a quite satisfactory recovery rate (~81 %). The limit of detection was 0.04 μg mL⁻¹, and the relative standard deviation was less than 2 %. Compared with conventional solid–liquid extraction, this new method avoids the use of volatile organic solvents, therefore is environmentally safer.

Keywords: Cannabis; Δ⁹-Tetrahydrocannabinol; Cloud point extraction; Coacervate; HPLC; Diode array detector; Nonionic surfactant

2-Methiopropamine, a thiophene analogue of methamphetamine: studies on its metabolism and detectability in the rat and human using GC-MS and LC-(HR)-MS techniques by Jessica Welter; Markus R. Meyer; Ehud (Udi) Wolf; Wolfgang Weinmann; Pierce Kavanagh; Hans H. Maurer (pp. 3125-3135).

2-Methiopropamine [1-(thiophen-2-yl)-2-methylaminopropane, 2-MPA], a thiophene analogue of methamphetamine, is available from online vendors selling “research chemicals.” The first samples were seized by the German police in 2011. As it is a recreational stimulant, its inclusion in routine drug screening protocols should be required. The aims of this study were to identify the phase I and II metabolites of 2-MPA in rat and human urine and to identify the human cytochrome-P450 (CYP) isoenzymes involved in its phase I metabolism. In addition, the detectability of 2-MPA in urine samples using the authors’ well-established gas chromatography–mass spectrometry (GC-MS) and liquid chromatography-linear ion trap-mass spectrometry (LC-MSⁿ) screening protocols was also evaluated. The metabolites were isolated from rat and human urine samples by solid-phase extraction without or following enzymatic cleavage of conjugates. The phase I metabolites, following acetylation, were separated and identified by GC-MS and/or liquid chromatography–high-resolution linear ion trap mass spectrometry (LC-HR-MSⁿ) and the phase II metabolites by LC-HR-MSⁿ. The following major metabolic pathways were proposed: N-demethylation, hydroxylation at the side chain and at the thiophene ring, and combination of these transformations followed by glucuronidation and/or sulfation. CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were identified as the major phase I metabolizing enzymes. They were also involved in the N-demethylation of the analogue methamphetamine and CYP2C19, CYP2D6, and CYP3A4 in its ring hydroxylation. Following the administration of a typical user’s dose, 2-MPA and its metabolites were identified in rat urine using the authors’ GC-MS and the LC-MSⁿ screening approaches. Ingestion of 2-MPA could also be detected by both protocols in an authentic human urine sample.

Keywords: Designer drugs; Methiopropamine; 2-MPA; Cytochrome-P450; GC-MS; LC-MS

Comparative study of recent wide-pore materials of different stationary phase morphology, applied for the reversed-phase analysis of recombinant monoclonal antibodies by Szabolcs Fekete; Jean-Luc Veuthey; Sebastiaan Eeltink; Davy Guillarme (pp. 3137-3151).

Various recent wide-pore reversed-phase stationary phases were studied for the analysis of intact monoclonal antibodies (mAbs) of 150 kDa and their fragments possessing sizes between 25 and 50 kDa. Different types of column technology were evaluated, namely, a prototype silica-based inorganic monolith containing mesopores of ∼250 Å and macropores of ∼ 1.1 μm, a column packed with 3.6 μm wide-pore core-shell particles possessing a wide pore size distribution with an average around 200 Å and a column packed with fully porous 1.7 μm particles having pore size of ∼300 Å. The performance of these wide-pore materials was compared with that of a poly(styrene–divinyl benzene) organic monolithic column, with a macropore size of approximately 1 μm but without mesopores (stagnant pores). A systematic investigation was carried out using model IgG1 and IgG2 mAbs, namely rituximab, panitumumab, and bevacizumab. Firstly, the recoveries of intact and reduced mAbs were compared on the two monolithic phases, and it appeared that adsorption was less pronounced on the organic monolith, probably due to the difference in chemistry (C18 versus phenyl) and the absence of mesopores (stagnant zones). Secondly, the kinetic performance was investigated in gradient elution mode for all columns. For this purpose, peak capacities per meter as well as peak capacities per time unit and per pressure unit (PPT) were calculated at various flow rates, to compare performance of columns with different dimensions. In terms of peak capacity per meter, the core-shell 3.6 μm and fully porous 1.7 μm columns outperformed the two monolithic phases, at a temperature of 60 °C. However, when considering the PPT values, the core-shell 3.6 μm column remained the best phase while the prototype silica-based monoliths became very interesting, mostly due to a very high permeability compared with the organic monolith. Therefore, these core-shell and silica-based monolith provided the fastest achievable separation. Finally, at the maximal working temperature of each column, the core-shell 3.6 μm column was far better than the other one, because it is the only one stable up to 90 °C. Lastly, the loading capacity was also measured on these four different phases. It appeared that the organic monolith was the less interesting and rapidly overloaded, due to the absence of mesopores. On the other hand, the loading capacity of prototype silica-based monolith was indeed reasonable.

Keywords: Monolith; Core-shell; Sub-2 μm; Column efficiency; Monoclonal antibody

Partial enzymatic elimination and quantification of sarcosine from alanine using liquid chromatography–tandem mass spectrometry by Casey Burton; Sanjeewa Gamagedara; Yinfa Ma (pp. 3153-3158).

Since sarcosine and d,l-alanine co-elute on reversed-phase high-performance liquid chromatography (HPLC) columns and the tandem mass spectrometer cannot differentiate them due to equivalent parent and fragment ions, derivatization is often required for analysis of sarcosine in LC/MS systems. This study offers an alternative to derivatization by employing partial elimination of sarcosine by enzymatic oxidation. The decrease in apparent concentration from the traditionally merged sarcosine–alanine peak associated with the enzymatic elimination has been shown to be proportional to the total sarcosine present (R ² = 0.9999), allowing for determinations of urinary sarcosine. Sarcosine oxidase was shown to eliminate only sarcosine in the presence of d,l-alanine, and was consequently used as the selective enzyme. This newly developed technique has a method detection limit of 1 μg/L (parts per billion) with a linear range of 3 ppb–1 mg/L (parts per million) in urine matrices. The method was further validated through spiked recoveries of real urine samples, as well as the analysis of 35 real urine samples. The average recoveries for low, middle, and high sarcosine concentration spikes were 111.7, 90.8, and 90.1 %, respectively. In conclusion, this simple enzymatic approach coupled with HPLC/MS/MS is able to resolve sarcosine from d,l-alanine leading to underivatized quantification of sarcosine. Figure Enzymatic elimination of sarcosine from alanine

Keywords: Sarcosine; Sarcosine oxidase; HPLC/MS/MS; Alanine; Enzymatic elimination

Newborn screening of phenylketonuria using direct analysis in real time (DART) mass spectrometry by Chunyan Wang; Hongbin Zhu; Zongwei Cai; Fengrui Song; Zhiqiang Liu; Shuying Liu (pp. 3159-3164).

Phenylketonuria (PKU) is commonly included in the newborn screening panel of most countries, with various techniques being used for quantification of l-phenylalanine (Phe). To diagnose PKU as early as possible in newborn screening, a rapid and simple method of analysis was developed. Using direct analysis in real time (DART) ionization coupled with triple-quadrupole tandem mass spectrometry (TQ-MS/MS) and with use of a 12 DIP-it tip scanner autosampler in positive ion mode, we analyzed dried blood spot (DBS) samples from PKU newborns. The concentration of Phe was determined using multiple reaction monitoring mode with the nondeuterated internal standard N,N-dimethylphenylalanine. The results of the analysis of DBS samples from newborns indicated that the DART-TQ-MS/MS method is fast, accurate, and reproducible. The results prove that this assay as a newborn screen for PKU can be performed in 18 s per sample for the quantification of Phe in DBS samples. DART-TQ-MS/MS analysis of the Phe concentration in DBS samples allowed us to screen newborns for PKU. This innovative protocol is rapid and can be effectively applied on a routine basis to analyze a large number of samples in PKU newborn screening and PKU patient monitoring. Figure The method can quantify the amount of phenylalanine in dried blood spot of newborn by using direct analysis in real time (DART) coupled with triple-quadrupole tandem mass spectrometry

Keywords: Direct analysis in real time (DART) triple-quadrupole tandem mass spectrometry; Phenylketonuria; Dried blood spot; Phenylalanine

Use of electrophoretic techniques and MALDI–TOF MS for rapid and reliable characterization of bacteria: analysis of intact cells, cell lysates, and “washed pellets” by Jiří Šalplachta; Anna Kubesová; Dana Moravcová; Marie Vykydalová; Sándor Süle; Hana Matoušková; Jaroslav Horký; Marie Horká (pp. 3165-3175).

In this study electrophoretic and mass spectrometric analysis of three types of bacterial sample (intact cells, cell lysates, and “washed pellets”) were used to develop an effective procedure for the characterization of bacteria. The samples were prepared from specific bacterial strains. Five strains representing different species of the family Rhizobiaceae were selected as model microorganisms: Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, R. galegae, R. loti, and Sinorhizobium meliloti. Samples of bacteria were subjected to analysis by four techniques: capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), gel IEF, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS). These methods are potential alternatives to DNA-based methods for rapid and reliable characterization of bacteria. Capillary electrophoretic (CZE and CIEF) analysis of intact cells was suitable for characterization of different bacterial species. CIEF fingerprints of “washed pellets” and gel IEF of cell lysates helped to distinguish between closely related bacterial species that were not sufficiently differentiated by capillary electrophoretic analysis of intact cells. MALDI–TOF MS of “washed pellets” enabled more reliable characterization of bacteria than analysis of intact cells or cell lysates. Electrophoretic techniques and MALDI–TOF MS can both be successfully used to complement standard methods for rapid characterization of bacteria.

Keywords: Bacteria; Characterization of bacteria; CZE; CIEF; IEF; MALDI

Miniaturised method for the quantitation of stabilisers in microtome cuts of polymer materials by HPLC with UV, MS or MS² detection by Martin Stiftinger; Wolfgang Buchberger; Christian W. Klampfl (pp. 3177-3184).

A method for quantitative depth profiling of polymer stabilisers in polypropylene materials is presented. Microtome cuts down to 10 μm thickness were prepared and an amount of as low as 10 mg was used for subsequent analysis. The sample preparation procedure included dissolution in toluene, followed by precipitation of the polymer by addition of methanol or acetonitrile, and subsequent analysis of the solution by reversed phase HPLC coupled with UV, MS and MS² detection. A comparison of the different detection techniques is given and their particular advantages are discussed. Depending on the stabiliser type, the presented method with MS detection can quantify stabiliser concentrations down to 0.007 mg L⁻¹ (this corresponds to 0.0007 mg g⁻¹ in the polymer sample) with repeatabilities better than 5 % relative standard deviation (n = 3). This equals a quantitation of absolute stabiliser amounts at the 0.1-μg level and concentrations down to 0.1 mg L⁻¹ in the corresponding polymer extracts. The developed method shows high sensitivity by the use of MS detection as well as good repeatabilities. Mainly due to the use of an appropriate internal standard, improved repeatability during sample extraction could be obtained. Furthermore, the applicability to real samples has been demonstrated.

Keywords: Polymer stabilisers; Microtome cuts; Miniaturised extraction method; High-performance liquid chromatography; Electrospray ionisation mass spectrometry

Quantitative LC–MS/MS method in urine for the detection of drugs used to reverse the effects of chemical castration by Sooyeun Lee; So-young Kang; Dajeong Ji; Seungkyung Baeck; Sangki Lee; Seung Min Oh; Kyu Hyuck Chung (pp. 3185-3194).

The chemical castration law, which targets child molesters with recidivism, was introduced in Korea in 2011. For this, leuprolide, a gonadotropin-releasing hormone agonist, is used to decrease testosterone production and suppress libido. In order to achieve efficient law enforcement, it is necessary to monitor intentional ingestion of drugs that antagonize the effect of leuprolide. Therefore, an analytical method for the simultaneous detection of mirodenafil, sildenafil, tadalafil, udenafil, vardenafil, icariin, alprostadil, and yohimbine, which are the major impotence treatment drugs, legitimately or otherwise, in Korea, as well as their selected metabolites, in human urine was established and validated using liquid chromatography–tandem mass spectrometry (LC–MS/MS). First, different sample preparation methods, two solid-phase extractions with different cartridges and protein precipitation, were compared and protein precipitation was chosen for the entire study because it showed better matrix effects and recoveries. Thus, the drugs and metabolites in urine were extracted by protein precipitation and then filtered and analyzed by LC–MS/MS with polarity switching electrospray ionization. The validation results of selectivity, matrix effect, recovery, linearity, intra- and inter-assay precision and accuracy were satisfactory. The limits of detection ranged from 0.25 to 10 ng/mL, and the limits of quantification were 2.5 to 50 ng/mL. The drugs and metabolites in urine did not show any degradation under storage for 7 and 15 days at 4 and −20 °C as well as after three freeze–thaw cycles. The developed method will be very useful for monitoring the illegal use of impotence treatment drugs.

Keywords: Chemical castration law; Impotence treatment drugs; Urine analysis; Matrix effects; LC–MS/MS

Simultaneous serum desalting and total protein determination by macroporous reversed-phase chromatography by Alexander Boichenko; Natalia Govorukhina; Ate G. J. van der Zee; Rainer Bischoff (pp. 3195-3203).

Macroporous reversed-phase (mRP) chromatography was successfully used to develop an accurate and precise method for total protein in serum. The limits of detection (0.83 μg, LOD) and quantification (2.51 μg, LOQ) for the mRP method are comparable with those of the widely used micro BCA protein assay. The mRP method can be used to determine the total protein concentration across a wide dynamic range by detecting chromatographic peaks at 215 nm and 280 nm. The method has the added advantage of desalting and denaturing proteins, leading to more complete digestion by trypsin and to better LC–MS–MS identification in shotgun proteomics experiments. Figure Simultaneous Serum Desalting and Total Protein Determination with Macroporous Reversed-Phase Chromatography: calibration plots

Keywords: Macroporous reversed-phase chromatography; Serum; Total protein concentration; Desalting; Digestion; LC–MS–MS; Proteomics

Rapid preparation of molecularly imprinted polymer by frontal polymerization by Dan-Dan Zhong; Xin Liu; Qian-Qian Pang; Yan-Ping Huang; Zhao-Sheng Liu (pp. 3205-3214).

Frontal polymerization was successfully applied, for the first time, to obtain molecularly imprinted polymers (MIPs). The method provides a solvent-free polymerization mode, and the reaction can be completed in 30 min. By this approach, MIPs were synthesized using a mixture of levofloxacin (template), methacrylic acid, and divinylbenzene. The effect of template concentration and the amount of comonomer on the imprinting effect of the resulting MIPs was investigated. The textural and morphological parameters of the MIP particles were also characterized by mercury intrusion porosimetry, nitrogen adsorption isotherms, and scanning electron microscopy, providing evidence concerning median pore diameter, pore volumes, and pore size distributions. The levofloxacin-imprinted polymer formed in frontal polymerization mode showed high selectivity, with an imprinting factor of 5.78. The results suggest that frontal polymerization provides an alternative means to prepare MIPs that are difficult to synthesize and may open up new perspectives in the field of MIPs. Figure ᅟ

Keywords: Frontal polymerization; Molecularly imprinted polymer; Molecular recognition; Affinity; Levofloxacin

LC-MS/TOF, LC-MSⁿ, on-line H/D exchange and LC-NMR studies on rosuvastatin degradation and in silico determination of toxicity of its degradation products: a comprehensive approach during drug development by Ravi P. Shah; Archana Sahu; Saranjit Singh (pp. 3215-3231).

The present study dealt with the forced degradation behaviour of rosuvastatin under ICH prescribed stress conditions. The drug was found to be labile under acid hydrolytic and photolytic conditions, while it was stable to base/neutral hydrolytic, oxidative and thermal stress. In total, 11 degradation products were formed, which were separated on a C-18 column using a stability-indicating method. LC-MS analyses indicated that five degradation products had the same molecular mass as that of the drug, while the remaining six had 18 Da less than the drug. Structure elucidation of all the degradation products was executed using sophisticated and modern structural characterization tools, viz. LC-MS/TOF, LC-MSⁿ, on-line H/D exchange and LC-NMR. The degradation pathway and mechanisms of degradation of the drug were delineated. Additionally, in silico toxicity was predicted for all the degradation products using TOPKAT and DEREK software and compared with the drug. This study demonstrates a comprehensive approach of degradation studies during the drug development phase. Figure Degradation pathway of rosuvastatin

Keywords: Rosuvastatin; Stress studies; Degradation product; LC-MS/TOF; MSⁿ studies; LC-NMR

Trace analysis of acetylcholinesterase inhibitors with antipsychotic drugs for Alzheimer’s disease by capillary electrophoresis with on column field-amplified sample injection by Yi-Rou Wang; Yuan-Han Yang; Chi-Yu Lu; Shun-Jin Lin; Su-Hwei Chen (pp. 3233-3242).

A simple and sensitive capillary zone electrophoresis (CZE) with UV detection (214 nm) was developed and validated for the simultaneous determination of the acetylcholinesterase inhibitors (AChEI), donepezil, and rivastigmine, with antipsychotic drugs in plasma. A sample pretreatment by liquid–liquid extraction and subsequent quantification by CZE with field-amplified sample injection (FASI) was used. The optimum separation for these analytes was achieved in <20 min at 25 °C with a fused-silica capillary column of 60.2 cm × 50 μm I.D. (effective length 50 cm) and a run buffer containing 120 mM phosphate (pH 4.0) with 0.1 % γ-cyclodextrin, 40 % methanol (MeOH), and 0.02 % polyvinyl alcohol as a dynamic coating to reduce analytes’ interaction with the capillary wall. Using phenformin as an internal standard (40.0 ng/mL), the linear ranges of the proposed method for the simultaneous determination of donepezil, rivastigmine, aripiprazole, quetiapine, risperidone, clozapine, ziprasidone, and trazodone were over the range 4.0–80.0 ng/mL, and olanzapine was over the range 1.0–20.0 ng/mL. The method was applied for concentrations monitoring of AChEIs and antipsychotic drugs in ten Alzheimer’s disease patients with behavioral and psychological symptoms of dementia after oral administration of the commercial products. Figure Electropherogram of tested chemicals in plasma determinations

Keywords: Determination of acetylcholinesterase inhibitors with antipsychotic drugs; Liquid–liquid extraction; Field-amplified sample injection; Alzheimer’s disease

The determination of nonylphenol and its precursors in a trickling filter wastewater treatment process by Bruce Petrie; Ewan J. McAdam; Mick J. Whelan; John N. Lester; Elise Cartmell (pp. 3243-3253).

An ultra performance liquid chromatography method coupled to a triple quadrupole mass spectrometer was developed to determine nonylphenol and 15 of its possible precursors (nonylphenol ethoxylates and nonylphenol carboxylates) in aqueous and particulate wastewater matrices. Final effluent method detection limits for all compounds ranged from 1.4 to 17.4 ng l⁻¹ in aqueous phases and from 1.4 to 39.4 ng g⁻¹ in particulate phases of samples. The method was used to measure the performance of a trickling filter wastewater treatment works, which are not routinely monitored despite their extensive usage. Relatively good removals of nonylphenol were observed over the biological secondary treatment process, accounting for a 53 % reduction. However, only an 8 % reduction in total nonylphenolic compound load was observed. This was explained by a shortening in ethoxylate chain length which initiated production of shorter polyethoxylates ranging from 1 to 4 ethoxylate units in length in final effluents. Modelling the possible impact of trickling filter discharge demonstrated that the nonylphenol environmental quality standard may be exceeded in receiving waters with low dilution ratios. In addition, there is a possibility that the EQS can be exceeded several kilometres downstream of the mixing zone due to the biotransformation of readily degradable short-chained precursors. This accentuates the need to monitor ‘non-priority’ parent compounds in wastewater treatment works since monitoring nonylphenol alone can give a false indication of process performance. It is thus recommended that future process performance monitoring and optimisation is undertaken using the full suite of nonylphenolic moieties which this method can facilitate.

Keywords: Surfactant; Alkylphenol; Carboxylate; UPLC; Model

Integrated procedure for multiresidue analysis of dissolved and particulate drugs in municipal wastewater by liquid chromatography–tandem mass spectrometry by Ivan Senta; Ivona Krizman; Marijan Ahel; Senka Terzic (pp. 3255-3268).

For the first time, an integrated procedure for a quantitative multiresidue analysis of dissolved and particulate illicit drug target residues was developed and validated in three different wastewater matrices. The procedure consists of a comprehensive sample enrichment, fractionation and cleanup followed by the determination of target analytes by triple quadrupole liquid chromatography–tandem mass spectrometry in both positive and negative ionisation polarities. The enrichment of illicit drugs from suspended solids and aqueous samples was performed using pressurised liquid extraction and solid phase extraction (SPE), respectively. The performance of different SPE cartridges was investigated in order to optimise the overall recovery and to reduce the matrix effects. The optimal results were obtained by combining mixed cation exchange (Oasis MCX) cartridges for fractionated enrichment, weak anion exchange for an additional extract cleanup and optimised chromatographic separation to minimise the impact from co-extracted interferences. The method was applied for the analysis of raw wastewater (RW), activated sludge (AS) and secondary effluent (SE) samples collected at four different wastewater treatment plants. The average contributions of the particulate drugs in the RW and AS were 1–28 and 23–65 %, respectively. This suggested that the total mass loads of some drugs might be underestimated by neglecting the particulate fraction. Moreover, relatively high distribution coefficients, determined for 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (RW = 1211 L/kg) and 11-hydroxy-Δ⁹-tetrahydrocannabinol (RW = 1,786 L/kg) implied that adsorption might play a significant role in their overall removal during wastewater treatment. Figure Scheme of the integrated analytical protocol for the analyses of particulate and dissolved drug target residues in wastewater

Keywords: Illicit drugs; Wastewater; LC-MS-MS; Dissolved drugs; Particulate drugs; Partitioning

Effervescence-assisted carbon nanotubes dispersion for the micro-solid-phase extraction of triazine herbicides from environmental waters by Guillermo Lasarte-Aragonés; Rafael Lucena; Soledad Cárdenas; Miguel Valcárcel (pp. 3269-3277).

Extraction techniques are surface-dependent processes since their kinetic directly depends on the contact area between the sample and the extractant phase. The dispersion of the extractant (liquid or solid) increases this area improving the extraction efficiency. In this article, the dispersion of a nanostructured sorbent at the very low milligram level is achieved by effervescence thanks to the in situ generation of carbon dioxide. For this purpose, a special tablet containing the effervescence precursors (sodium carbonate as carbon dioxide source and sodium dihydrogen phosphate as proton donor) and the sorbent [multiwalled carbon nanotubes (MWCNTs)] is prepared. All the microextraction steps take place in a glass beaker containing 100 mL of the sample. After the extraction, the MWCNTs, enriched with the extracted analytes, are recovered by vacuum filtration. Methanol was selected to elute the retained analytes. The extraction mode is optimized and characterized using the determination of nine herbicides in water samples as model analytical problem. The absolute recoveries of the analytes were in the range 48–76 %, while relative recoveries were close to 100 % in all cases. These values permit the determination of these analytes at the low microgram per liter range with good precision (relative standard deviations lower than 9.3 %) using ultra performance liquid chromatography (UPLC) combined with ultraviolet detection (UV).

Keywords: Effervescence-assisted dispersion; Multiwalled carbon nanotubes; Triazines; Water samples

Hybrid flow analyzer for automatic hollow-fiber-assisted ionic liquid-based liquid-phase microextraction with in-line membrane regeneration by Sira Nitiyanontakit; Pakorn Varanusupakul; Manuel Miró (pp. 3279-3288).

The proof-of-concept of a new methodology for in-line hollow-fiber (HF)-assisted three-phase liquid-phase microextraction (LPME) allowing for handling of the feed and acceptor aqueous solutions and of minute volumes of the organic extracting phase in a programmable flow mode is reported in this paper. The flow analyzer fosters in-line anchoring of ionic-liquid-laden extracting solution (10 % (v/v) methyltrioctyl ammonium chloride in kerosene) in the pores of a single-strand microporous polypropylene HF, and regeneration of the liquid-phase membrane itself for each individual analysis cycle in a fully automated mode. Using hexavalent chromium as a model analyte and 1,5-diphenylcarbazide as a chromogenic probe in the acceptor solution, the flow-based HF-LPME hyphenated system was harnessed to the clean-up of troublesome samples (viz., domestic wastewater and soil leachates) with concomitant enrichment of target species. Distinct extraction modes and chemistries were assessed for enhanced Cr(VI) permeability. A single sample plug was subjected to a twofold backward–forward flow extraction so as to decrease the thickness of the boundary layer at the HF shell side for improved extraction efficiency. Under the optimized physicochemical variables, a limit of detection of 4.6 μg L⁻¹ Cr(VI), a dynamic linear range of up to 500 μg L⁻¹ and intermediate precision better than 10 % were obtained for a sample volume of 2.8 mL buffered at pH 4 and a volume of organic extractant of 120 μL, with an enrichment factor of ca. 11 for a sample residence time in the donor compartment of merely 4.5 min. Analyte recoveries in domestic wastewaters were ≥83 % using external calibration with relative standard deviations better than 14 %, thereby demonstrating the expedient clean-up of samples with elevated content of dissolved organic carbon. The automatic HF-LPME method was validated in terms of bias against the SRM 2701 (NIST soil) preceded by the EPA alkaline digestion method 3060A. No significant differences between Cr(VI) concentration as obtained with the automatic HF-LPME system (546 ± 57 mg kg⁻¹) and the certified value (551.2 ± 17.2 mg kg⁻¹, expressed as mean ± combined uncertainty) were encountered at the 0.05 significance level.

Keywords: Automation; Supported liquid membrane; Ionic liquid; Miniaturization; In-line membrane regeneration

The use of mass defect plots for the identification of (novel) halogenated contaminants in the environment by Karl J. Jobst; Li Shen; Eric J. Reiner; Vince Y. Taguchi; Paul A. Helm; Robert McCrindle; Sean Backus (pp. 3289-3297).

Mass defect is the difference between the nominal and exact mass of a chemical element or compound. An intrinsic property of polyhalogenated molecules is a uniquely negative mass defect, which readily distinguishes halogenated from non-halogenated compounds in a complex mass spectrum and can be visualized by constructing a mass defect plot. This study demonstrates the utility of the mass defect plot as a powerful tool to screen gas-chromatography (ultra)high-resolution mass spectrometry data for potentially toxic and bioaccumulative halogenated compounds in a Lake Ontario lake trout, an apex species in the Great Lakes environment. Our results indicate that the sample is contaminated with polychlorinated biphenyls, terphenyls, diphenylethers, as well as other chlorinated pesticides and flame retardants that are regulated and routinely analyzed by traditional target analyses. However, the mass defect plot also displays peaks which could be traced to the presence of as yet undiscovered contaminants. These include chlorinated polycyclic aromatic hydrocarbons as well as mixed halogenated analogues of the flame retardant Dechlorane 604. The identity of the latter class of compounds is supported by experiments with genuine standards. Fig The mass defect plot provides an informative picture of the halogenated contaminants in a sample of Lake Ontario lake trout.

Keywords: Mass defect plot; Kendrick plot; FT-ICR; High-resolution mass spectrometry; Halogenated flame retardants; Dechlorane 604; Chlorinated PAH

Influence of heteroatom pre-selection on the molecular formula assignment of soil organic matter components determined by ultrahigh resolution mass spectrometry by Tsutomu Ohno; Paul E. Ohno (pp. 3299-3306).

Soil organic matter (SOM) is involved in many important ecosystem processes. Ultrahigh resolution mass spectrometry has become a powerful technique in the chemical characterization of SOM, allowing assignment of elemental formulae for thousands of peaks resolved in a typical mass spectrum. We investigated how the addition of N, S, and P heteroatoms in the formula calculation stage of the mass spectra processing workflow affected the formula assignments of mass spectra peaks. Dissolved organic matter extracted from plant biomass and soil as well as the soil humic acid fraction was studied. We show that the addition of S and P into the molecular formula calculation increased peak assignments on average by 17.3 % and 10.7 %, respectively, over the assignments based on the CHON elements frequently reported by SOM researchers using ultrahigh resolution mass spectrometry. The organic matter chemical characteristics as represented by van Krevelen diagrams were appreciably affected by differences in the heteroatom pre-selection for the three organic matter samples investigated, especially so for the wheat-derived dissolved organic matter. These results show that inclusion of both S and P heteroatoms into the formula calculation step, which is not routinely done, is important to obtain a more chemically complete interpretation of the ultrahigh resolution mass spectra of SOM.

Keywords: Dissolved organic matter (DOM); Ion cyclotron resonance mass spectrometry; Molecular formula calculation; Humic acid

Mixed immunoassay design for multiple chemical residues detection by Yanshen Li; Peng Li; Xiangshu Luo; Zhihui Hao; Zhanhui Wang; Jianzhong Shen; Xingyuan Cao; Suxia Zhang (pp. 3307-3312).

In this research, a mixed immunoassay design for multiple chemical residues detection based on combined reverse competitive enzyme-linked immunosorbent assay (ELISA) procedure was developed. This method integrated two reverse ELISA reactions in one assay by labeling horseradish peroxidase to deoxynivalenol (DON) and orbifloxacin. Within this method, IC50 of the two mAbs for each analyte we produced ranged from 23 ∼ 68 ng mL⁻¹ for DONs and 4.1 ∼ 49 ng mL⁻¹ for quinolones (QNs). The limit of detection measured by IC10 was achieved at 0.45–1.3 ng mL⁻¹ for DONs and 0.59–6.9 ng mL⁻¹ for QNs, which was lower than the maximum residue levels. Recoveries in negative samples spiked at concentrations of 100, 200, and 500 ng mL⁻¹ ranged from 91.3 to 102.2 % for DONs and 88.7–98.05 % for QNs with relative standard deviation less than 9.88 and 12.67 %. The results demonstrated that this developed immunoassay was suitable for screening of low molecular weight contaminants. Figure Combined reverse ELISA procedure for multi-chemical residues analysis

Keywords: Deoxynivalenol; Quinolone; Analysis; Integrated enzyme-linked immunosorbent assay

Lateral-flow enzyme immunoconcentration for rapid detection of Listeria monocytogenes by Il-Hoon Cho; Joseph Irudayaraj (pp. 3313-3319).

Lateral-flow enzyme immunochromatography coupled with an immunomagnetic step was developed for rapid detection of Listeria monocytogenes in food matrices. The target bacteria was first separated and concentrated by magnetic nanoparticles containing the enzyme and directly applied to the assay system to induce an antigen–antibody reaction without any additional steps. The color signals produced by an enzyme–substrate reaction at a specific site on the immunostrip were found to be directly proportional to the concentration of L. monocytogenes in the sample. The detection concept was demonstrated by performing an enzyme immunoassay on a microtiter well prior to applying it to the lateral-flow assay. Results of the chromatographic analysis yield a limit of detection of 95 and 97 ± 19.5 CFU/mL in buffer solution and 2 % milk sample, respectively. In addition to the high sensitivity, it was also possible to shorten the separation and detection time to within 2 h. The system also showed no cross-reactivity with other bacteria (e.g., Escherichia coli O157:H7, Salmonella typhimurium, and Salmonella enteritidis). The analytical procedure developed will enable us to not only utilize the assay in the field where fast screening for pathogenic agents is required but also as a preventive measure to contain disease outbreak.

Keywords: Listeria monocytogenes ; Lateral flow; Immunochromatography; Enzyme immunoassay; Signal enhancement; Immunomagnetic concentration

Identification of metabolic profiling of cell culture of licorice compared with its native one by Shuli Man; Songbo Guo; Wenyuan Gao; Juan Wang; Liming Zhang; Xinglin Li (pp. 3321-3329).

Glycyrrhiza uralensis has long been used as a flavoring and sweetening agent in food products. In the last ten years, suspensions of Glycyrhiza cells have been successfully established. However, there is no report of full metabolic profiling research on these cells. To identify their composition we used HPLC–DAD coupled with ESI(+/−)–MS n to compare the constituents of cultured Glycyrhiza (CG) cells with those the native cells (NG). We identified 60 compounds including flavonoids, phenols, and triterpenoids. Among these compounds, 42 occurred both in NG and CG, nine were present in NG only and nine were present in CG alone. The number of the triterpenoid aglycones without glycones in CG was smaller than that in NG. The number of flavanone, isoflavone, isoflavan, and benzenoid compounds was also smaller in CG than that in NG, whereas the number of pterocarpans was much higher. Although differences existed between CG and NG, the extract of CG was similar to that of NG. With the development of cell suspension culture-based biotransformation, cell culture of Glycyrrhiza has the potential to be more profitable than field cultivation in some areas.

Keywords: Glycyrrhiza; Suspension cell; Field cultivation; Metabolic profiling; LC–MS n

Simultaneous quantification of Pacific ciguatoxins in fish blood using liquid chromatography–tandem mass spectrometry by Yim Ling Mak; Jia Jun Wu; Wing Hei Chan; Margaret B. Murphy; James C. W. Lam; Leo L. Chan; Paul K. S. Lam (pp. 3331-3340).

Ciguatera fish poisoning (CFP) is a food intoxication caused by exposure to ciguatoxins (CTXs) in coral reef fish. Rapid analytical methods have been developed recently to quantify Pacific-CTX-1 (P-CTX-1) in fish muscle, but it is destructive and can cause harm to valuable live coral reef fish. Also fish muscle extract was complex making CTX quantification challenging. Not only P-CTX-1, but also P-CTX-2 and P-CTX-3 could be present in fish, contributing to ciguatoxicity. Therefore, an analytical method for simultaneous quantification of P-CTX-1, P-CTX-2, and P-CTX-3 in whole blood of marketed coral reef fish using sonication, solid-phase extraction (SPE), and liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. The optimized method gave acceptable recoveries of P-CTXs (74–103 %) in fish blood. Matrix effects (6–26 %) in blood extracts were found to be significantly reduced compared with those in muscle extracts (suppressed by 34–75 % as reported in other studies), thereby minimizing potential for false negative results. The target P-CTXs were detectable in whole blood from four coral reef fish species collected in a CFP-endemic region. Similar trends in total P-CTX levels and patterns of P-CTX composition profiles in blood and muscle of these fish were observed, suggesting a relationship between blood and muscle levels of P-CTXs. This optimized method provides an essential tool for studies of P-CTX pharmacokinetics and pharmacodynamics in fish, which are needed for establishing the use of fish blood as a reliable sample for the assessment and control of CFP. Figure Chromatograph of P-CTXs in whole blood of wild-caught moray eel

Keywords: Ciguatera fish poisoning; Pacific ciguatoxins; P-CTXs; Fish blood; LC-MS/MS

Spatially resolved (semi)quantitative determination of iron (Fe) in plants by means of synchrotron micro X-ray fluorescence by Roberto Terzano; Matthias Alfeld; Koen Janssens; Bart Vekemans; Tom Schoonjans; Laszlo Vincze; Nicola Tomasi; Roberto Pinton; Stefano Cesco (pp. 3341-3350).

Iron (Fe) is an essential element for plant growth and development; hence determining Fe distribution and concentration inside plant organs at the microscopic level is of great relevance to better understand its metabolism and bioavailability through the food chain. Among the available microanalytical techniques, synchrotron μ-XRF methods can provide a powerful and versatile array of analytical tools to study Fe distribution within plant samples. In the last years, the implementation of new algorithms and detection technologies has opened the way to more accurate (semi)quantitative analyses of complex matrices like plant materials. In this paper, for the first time the distribution of Fe within tomato roots has been imaged and quantified by means of confocal μ-XRF and exploiting a recently developed fundamental parameter-based algorithm. With this approach, Fe concentrations ranging from few hundreds of ppb to several hundreds of ppm can be determined at the microscopic level without cutting sections. Furthermore, Fe (semi)quantitative distribution maps were obtained for the first time by using two opposing detectors to collect simultaneously the XRF radiation emerging from both sides of an intact cucumber leaf. Figure Elemental distribution maps within intact tomato roots as determined by confocal micro X‐ray fluorescence

Keywords: Iron; Synchrotron; X-ray fluorescence; Quantitative analysis; Tomato; Cucumber

Fluorescence properties of Ca²⁺-independent discharged obelin and its application prospects by Roza R. Alieva; Nadezhda V. Belogurova; Alena S. Petrova; Nadezhda S. Kudryasheva (pp. 3351-3358).

Discharged obelin, a complex of coelenteramide and polypeptide, is a fluorescent protein produced from the photoprotein obelin, which is responsible for bioluminescence of the marine hydroid Obelia longissima. Discharged obelin is stable and nontoxic and its spectra are variable, and this is why it can be used as a fluorescent biomarker of variable color in vivo and in vitro. Here we examined light-induced fluorescence of Ca²⁺-independent discharged obelin (obtained without addition of Ca²⁺). Its emission and excitation spectra were analyzed under variation of the excitation wavelength (260–390 nm) and the emission wavelength (400–700 nm), as well as the 40 °C exposure time. The emission spectra obtained with excitation at 260–300 nm (tryptophan absorption region) included three peaks with maxima at 355, 498, and 660 nm, corresponding to fluorescence of tryptophan, polypeptide-bound coelenteramide, and a hypothetical indole–coelenteramide exciplex, respectively. The emission spectra obtained with excitation at 310–380 nm (coelenteramide absorption region) did not include the 660-nm maximum. The peak in the red spectral region (λ _max = 660 nm) has not been previously reported. Exposure to 40 °C under excitation at 310–380 nm shifted the obelin fluorescence spectra to the blue, whereas excitation at 260–300 nm shifted them to the red. Hence, red emission and variation of the excitation wavelength form a basis for development of new medical techniques involving obelin as a colored biomarker. The addition of red color to the battery of known (violet to yellow) colors increases the potential of application of obelin.

Keywords: Fluorescent protein; Discharged photoprotein obelin; Fluorescence color; Thermoinactivation; Excitation wavelength; Proton transfer

²⁹Si{¹H} CP-MAS NMR comparison and ATR-FTIR spectroscopic analysis of the diatoms Chaetoceros muelleri and Thalassiosira pseudonana grown at different salinities by Sian M. La Vars; Martin R. Johnston; John Hayles; Jason R. Gascooke; Melissa H. Brown; Sophie C. Leterme; Amanda V. Ellis (pp. 3359-3365).

Diatoms are key indicators of marine environmental health. To further understand how diatoms respond to varying degrees of salinity, either due to climate change or brine waste discharge into marine environments, two different diatom species were studied. Thalassiosira pseudonana and Chaetoceros muelleri were cultured at three different salinities namely, 26 practical salinity units (PSU or parts per thousand), 36 PSU (standard salinity for culturing of seawater species) and 46 PSU. Changes in silica and organic content within the cultured diatoms were analysed using solid-state ²⁹Si{¹H} cross-polarization–magic angle spinning (CP-MAS) nuclear magnetic resonance (NMR) and attenuated total reflection–Fourier transform infrared (ATR-FTIR) spectroscopies coupled with analysis of variance. ²⁹Si CP-MAS NMR showed that qualitatively the Q4:Q3 area ratios of C. muelleri, grown away from standard salinities, increased in response to the formation of more condensed (2 ≡SiOH → ≡Si–O–Si≡ + H₂O) and/or an increase in closely associated organic matter to the Q4 component of the diatoms. This was not observed for T. pseudonana. However, both species showed the appearance of a new peak centered at 1575–1580 cm⁻¹ in the ATR-FTIR spectra, designated as the C═N band of nitrogenous purine-type compounds. Further, the C. muelleri species was shown to produce more extracellular polymeric substances at non-standard salinities. On this basis, results suggest that there is a strong relationship between diatom composition and salinity and that C. muelleri is more sensitive to its environment than T. pseudonana.

Keywords: Diatoms; Salinity; ²⁹Si solid-state nuclear magnetic resonance; Fourier transform infrared spectroscopy

Quantitative Raman spectroscopy in turbid matter: reflection or transmission mode? by Dieter Oelkrug; Edwin Ostertag; Rudolf W. Kessler (pp. 3367-3379).

Raman intensities from reflection (X R) and transmission (X T) setups are compared by calculations based on random walk and analytical approaches with respect to sample thickness, absorption, and scattering. Experiments incorporating strongly scattering organic polymer layers and powder tablets of pharmaceutical ingredients validate the theoretical findings. For nonabsorbing layers, the Raman reflection and transmission intensities rise steadily with the layer thickness, starting for very thin layers with the ratio X T/X R = 1 and approaching for thick layers, a lower limit of X T/X R = 0.5. This result is completely different from the primary irradiation where the ratio of transmittance/reflectance decays hyperbolically with the layer thickness to zero. In absorbing materials, X R saturates at levels that depend strongly on the absorption and scattering coefficients. X T passes through a maximum and decreases then exponentially with increasing layer thickness to zero. From the calculated radial intensity spreads, it follows that quantitative transmission Raman spectroscopy requires diameters of the detected sample areas be about six times larger than the sample thickness. In stratified systems, Raman transmission allows deep probing even of small quantities in buried layers. In double layers, the information is independent from the side of the measurements. In triple layers simulating coated tablets, the information of X _T originates mainly from the center of the bulk material whereas X R highlights the irradiated boundary region. However, if the stratified sample is measured in a Raman reflection setup in front of a white diffusely reflecting surface, it is possible to monitor the whole depth of a multiple scattering sample with equal statistical weight. This may be a favorable approach for inline Raman spectroscopy in process analytical technology. Figure Raman spectroscopy in turbid matter

Keywords: Reflection and transmission Raman spectroscopy; Diffuse light scattering; Process analytical technology (PAT); Pharmaceutical analysis; Monte Carlo simulations; Random walk

Study of metabonomic profiles of human esophageal carcinoma by use of high-resolution magic-angle spinning ¹H NMR spectroscopy and multivariate data analysis by Yongxia Yang; Lijing Wang; Shumei Wang; Shengwang Liang; Ali Chen; Huiru Tang; Lei Chen; Feng Deng (pp. 3381-3389).

Esophageal carcinoma (EC) is one of the most common malignant tumors. EC survival has remained disappointingly low because of the high malignancy of esophageal cancer and the lack of obvious clinical symptoms at an early stage. Early diagnosis is often difficult because the small tumor nodules are frequently missed. Metabonomics based on high-resolution magic-angle spinning (HRMAS) NMR has been popular for tumor detection because it is highly sensitive, provides rich biochemical information and requires no sample pretreatment. ¹H HRMAS spectra of non-involved adjacent esophageal tissues and of well differentiated and moderately differentiated esophageal carcinoma tumors were recorded and analyzed by use of multivariate and statistical analysis techniques. Moderately differentiated EC tumors were found to have increased total choline, alanine, and glutamate and reduced creatine, myo-inositol, and taurine compared with non-involved adjacent tissues. Moreover, clear differences between the metabonomic profiles of EC tissues enabled tumor differentiation. Furthermore, the integral Gly/MI ratio for samples of different tissue types were statistically significantly different; this was sufficient both for distinguishing non-involved tissues from esophageal carcinoma and for classification of well differentiated and moderately differentiated EC tumors. Figure Tissue metabonomics analysis based on the HRMAS ¹H NMR spectroscopy is a powerful nondestructive approach in characterizing the metabolite composition in human esophageal carcinoma (EC), in the development of new diagnostic methods, and perhaps in the evaluation processes of clinical therapies. The result demonstrated that (a) the metabonomes of both well-differentiated EC and moderately differentiated EC tumors differ markedly from that of the adjacent non-involved tissues, and (b) well-differentiated EC tumors have clear differences in metabonome from that of the moderately differentiated EC tumors by using multivariate data analysis

Keywords: High-resolution magic-angle spinning spectroscopy; ¹H NMR; Esophageal carcinoma; Metabonome

Capillary electrophoretic study of the synergistic biological effects of alkaloids from Chelidonium majus L. in normal and cancer cells by Maria Kulp; Olga Bragina (pp. 3391-3397).

In this study, the synergistic biological action of five celandine alkaloids in normal and cancer cells was investigated by capillary electrophoresis with light-emitting diode-induced native fluorescence detection. The specific capacity of each alkaloid to penetrate into the cells was estimated by monitoring alkaloid concentration decreases in the cell medium during incubation with murine fibroblast NIH/3T3, mouse melanoma B16F10, and human breast cancer MCF7 cell lines. Mixtures of isoquinoline alkaloids containing protopine, chelidonine, sanguinarine, allocryptopine, and stylopine were applied to cell cultures for 20 and 40 min, and the content of alkaloids in the cell media was measured by capillary electrophoresis (CE). CE separation of isoquinoline alkaloids was performed in 30 mM phosphate buffer (pH 2.5). As these alkaloids have native fluorescence, they were directly detected using the commercially available UV light-emitting diode without troublesome fluorescent derivatization. The results showed a differential ability of celandine alkaloids to penetrate into the normal and cancer cell interior, which was inversely proportional to their cytotoxic activity. While the most effective transport of celandine alkaloids from the cell medium to the cell interior was observed for normal murine fibroblast NIH/3T3 cells (about 55% of total content), cytotoxicity tests demonstrated selective and profound apoptotic effects of a five-alkaloid combination in the mouse melanoma B16F10 cell line.

Keywords: Isoquinoline alkaloids; Cytotoxicity; Capillary electrophoresis; LED fluorescence detection

Separation of dansylated 17β-estradiol, 17α-estradiol, and estrone on a single HPLC column for simultaneous quantitation by LC–MS/MS by Szabolcs Szarka; Vien Nguyen; Laszlo Prokai; Katalin Prokai-Tatrai (pp. 3399-3406).

We show here that baseline separation of dansylated estrone, 17β-estradiol, and 17α-estradiol can be done, contrary to previous reports, within a short run time on a single RP-LC analytical column packed with particles bonded with phenyl-hexyl stationary phase. The chromatographic method coupled with isotope dilution tandem MS offers a simple assay enabling the simultaneous analysis of these analytes. The method employs ¹³C-labeled estrogens as internal standards to eliminate potential matrix effects arising from the use of deuterated estrogens. The assay also offers adequate accuracy and sensitivity to be useful for biological samples. The practical applicability of the validated method is demonstrated by the quantitative analyses of in vivo samples obtained from rats treated with Premarin®. Figure Quantification of estrogens from rat samples by LC–MS/MS

Keywords: Estrogens; Phenyl-hexyl stationary phase; LC–MS/MS; Dansylation; ¹³C-labeled internal standards

A 3-D open-framework material with intrinsic chiral topology used as a stationary phase in gas chromatography by Sheng-Ming Xie; Xin-Huan Zhang; Ze-Jun Zhang; Mei Zhang; Jia Jia; Li-Ming Yuan (pp. 3407-3412).

Compared with liquid chromatography and capillary electrophoresis, the diversity of gas chromatography chiral stationary phases is rather limited. Here, we report the fabrication of Co(d-Cam)_1/2(bdc)_1/2(tmdpy) (d-Cam = d-camphoric acid; bdc = 1,4-benzenedicarboxylate; tmdpy = 4,4′-trimethylenedipyridine)-coated open tubular columns for high-resolution gas chromatographic separation of compounds. The Co(d-Cam)_1/2(bdc)_1/2(tmdpy) compound possesses a 3-D framework containing enantiopure building blocks embedded in intrinsically chiral topological nets. In this study, two fused-silica open tubular columns with different inner diameters and lengths, including column A (30 m × 530 μm i.d.) and column B (2 m × 75 μm i.d.), were prepared by a dynamic coating method using Co-(d-Cam)_1/2(bdc)_1/2(tmdpy) as the stationary phase. The chromatographic properties of the two columns were investigated using n-dodecane as the test compound at 120 °C. The number of theoretical plates (plates/m) of the two metal–organic framework columns was 1,450 and 3,100, respectively. The separation properties were evaluated using racemates, isomers, alkanes, alcohols, and Grob's test mixture. The limit of detection and limit of quantification were found to be 0.125 and 0.417 ng for citronellal enantiomers, respectively. Repeatability (n = 6) showed lower than 0.25 % relative standard deviation (RSD) for retention times and lower than 2.2 % RSD for corrected peak areas. The experimental results showed that the stationary phase has excellent selectivity and also possesses good recognition ability toward these organic compounds, especially chiral compounds. Figure ᅟ

Keywords: Metal–organic frameworks; Open tubular columns; Stationary phase; Gas chromatography; Chiral topology; Chiral separation

Purification of saponins from leaves of Panax notoginseng using preparative two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography by Xiujie Guo; Xiuli Zhang; Jiatao Feng; Zhimou Guo; Yuansheng Xiao; Xinmiao Liang (pp. 3413-3421).

Saponins are widely distributed in the plant kingdom and have been shown to be active components of many medicinal herbs. In this study, a two-dimensional purification method based on reversed-phase liquid chromatography coupled with hydrophilic interaction liquid chromatography was successfully applied to purify saponins from leaves of Panax notoginseng. Nine saponin reference standards were used to test the separation modes and columns. The standards could not be resolved using C₁₈ columns owing to their limited polar selectivity. However, they were completely separated on a XAmide column in hydrophilic interaction liquid chromatography mode, including two pairs of standards that were coeluted on a C₁₈ column. The elution order of the standards on the two columns was sufficiently different, with a correlation coefficient between retention times on the C₁₈ and XAmide columns of 0.0126, indicating good column orthogonality. Therefore, the first-dimension preparation was performed on a C₁₈ column, followed by a XAmide column that was used to separate the fractions in the second dimension. Fifty-four fractions were prepared in the first dimension, with 25 fractions rich in saponins. Eight saponins, including two pairs of isomeric saponins and one new saponin, were isolated and identified from three representative fractions. This procedure was shown to be an effective approach for the preparative isolation and purification of saponins from leaves of P. notoginseng. Moreover, this method could possibly be employed in the purification of low-content and novel active saponins from natural products. Figure Separation of saponins using 2-D RPLC/HILIC

Keywords: Saponins; Purification; Reversed-phase liquid chromatography; Hydrophilic interaction chromatography; Panax notoginseng (Burk.) F.H.Chen

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Analytical and Bioanalytical Chemistry (v.405, #10)