Analytical Methods (v.10, #27)

Front cover (3295-3296).

Contents list (3297-3302).

This guide is designed for those involved in the purchase of analytical instrumentation, including analysts and laboratory staff, purchasing officers and senior managers. It provides recommendations on the stages that are required for the successful evaluation, purchase, installation and reliable operation of analytical instrumentation. The overall intention is to recommend a process where an analyst can make a confident recommendation of instrumentation that meets the desired specification. In addition, guidance is offered to ensure clarity in the roles and responsibilities of those involved in the overall procurement of a major analytical instrument.

Direct online quantitation of 2-methyl-3-methoxy-4-phenyl butanoic acid for total microcystin analysis by condensed phase membrane introduction tandem mass spectrometry by Kyle D. Duncan; Daniel G. Beach; Elliott J. Wright; Todd Barsby; Chris G. Gill; Erik T. Krogh (3310-3316).
Microcystins (MCs) are a potent class of hepatotoxins produced by several species of cyanobacteria, which have been responsible for illness and death in livestock and humans. Rapid detection and quantitation are important for the protection of human and environmental health. More than 200 MC variants have been reported, and nearly all release 2-methyl-3-methoxy-4-phenyl butanoic acid (MMPB) upon oxidation. The quantitation of MMPB directly in complex post-oxidation solutions is presented using online condensed phase membrane introduction mass spectrometry with a methanol acceptor phase and negative electrospray ionization. We observe a linear response over several orders of magnitude for MMPB standards, and have applied the technique to measuring total MC in algal suspensions with both positive and negative controls. Preliminary results are reported for the analysis of total microcystins after Lemieux oxidation, at detection limits relevant to regulatory levels in recreational and drinking waters, with no sample clean up. Good quantitative agreement was obtained between measurement of total MCs in an algal reference material by CP-MIMS-MS/MS and LC-MS/MS analysis of total and free MCs. The developed methodology is of interest as an important first step for further development as a rapid benchtop tool for quantitative analysis with future potential use in the field.

Magnetic surface molecularly imprinted polymeric microspheres using gallic acid as a segment template for excellent recognition of ester catechins by Bin Wang; Huiyun Deng; Mao Wu; Shu Xiang; Qiang Ma; Shuyun Shi; Lianwu Xie; Yaping Guo (3317-3324).
Magnetic surface molecularly imprinted polymeric microspheres (MMIPs) were prepared by using gallic acid (GA) as a segment template for the solid extraction of ester catechins as a whole from complex samples. MMIPs were characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and vibrating sample magnetometry. The results of the adsorption experiments indicated that the saturated adsorption capacity of GA on MMIPs was 0.21 mmol g−1, and the imprinting factor was 5.86; MMIPs had higher adsorption capacity for ester catechins than benzoic acid, salicylic acid, and 2,4-dihydroxybenzoic acid. The adsorption process of MMIPs could be better described by pseudo secondary rate equation, and it was consistent with the Langmuir adsorption model. In addition to GA, MMIPs could selectively recognize ester catechins in a tea solution without column packing. Thus, MMIPs prepared using GA as a segment template can be used in selective separation and enrichment of ester catechins from tea samples for functional food production.

An early fire sensor based on infrared gas analytical methods by Jingmin Dang; Haiye Yu; Chuantao Zheng; Yiding Wang; Yujing Sun (3325-3331).
Fire is the rapid oxidation reaction of a material in the chemical process of combustion, which releases heat, light, and various reaction products. Fires account for huge numbers of casualties and economic losses each year in spite of the widespread use of fire sensors. In comparison to traditional smoke or temperature-based analytical methods, the gas sensor-based methods are more effective since gases are produced in every fire while not all fires emit smoke aerosols during the combustion process, and the changes of ambient temperature in the early stage of a fire are too small to be sensed. Among the fire gases, carbon monoxide (CO), which has trace concentration levels (down to 0.1 parts per million by volume (ppmv)) in ambient air but is produced at high levels (up to vol%) in smoldering fires, is a promising gas for early fire detection. In this study, a laser-based sensor has been developed for the high precision and highly sensitive measurement of CO produced by fires. The sensor relies on a continuous wave (CW), distributed feedback (DFB) laser emitting at ∼2.33 μm as the excitation source. A wavelength modulation spectroscopy (WMS)-2f/WMS-1f strategy was adopted to isolate complex, overlapping spectral absorption features at ambient pressures and to achieve excellent specificity and high detection sensitivity. The long-term performance of the sensor system was evaluated by the Allan–Werle deviation analysis. The results showed that the limit of detection (LoD) and the optimal integration time are 1.18 ppmv and ∼205 s (corresponding to a measurement precision of ∼0.08 ppmv on average), respectively. As a field application, our sensor was used for the early detection of fires from paper, cotton and pine wood, which verified its reliable and robust operation.

Separation and detection of mutans streptococci by using magnetic nanoparticles stabilized with a cell wall binding domain-conjugated polymer by Panida Thanyasrisung; Aemvika Vittayaprasit; Oranart Matangkasombut; Motoyuki Sugai; Piyaporn Na Nongkai; Suttawan Saipia; Voravee P. Hoven (3332-3339).
A number of salivary mutans streptococci (MS: Streptococcus mutans and Streptococcus sobrinus) are used in dental caries risk assessment. In this study, a simple, yet effective assay was developed for MS detection. Magnetic nanoparticles (MNPs) were first grafted with poly(acrylic acid) that bears active carboxyl groups available for conjugation with the cell wall binding domain (CWBD) of automutanolysin which specifically binds to MS. The binding efficiency of CWBD-conjugated MNPs to MS was tested with pure cultures of streptococcal standard strains. After mixing CWBD-conjugated MNPs with culture, bacteria-bound particles were separated from unbound cells using a magnet and filtered through a cellulose acetate membrane (pore-size 0.8 μm). The color intensity of particles remaining on the membrane represents the number of bound bacteria. The CWBD-conjugated MNPs showed higher efficiency in binding to S. mutans and S. sobrinus than to non-mutans streptococci (S. sanguinis and S. salivarius) with capture efficiencies of 77 and 69% for MS and 38 and 15% for non-MS. Moreover, this method can quantify the number of MS in the range of 102 to 107 colony-forming units (CFU) mL−1, which covers the range of MS levels used in caries risk assessment. The calculated limit of detection of the assay was 16 and 72 CFU mL−1 for S. mutans and S. sobrinus, respectively. Furthermore, the CWBD-conjugated MNPs could be used to efficiently quantify the number of MS in human saliva samples containing highly complex mixtures of bacterial species. These results suggest that the assay could be applicable as a simple tool for MS determination in not only clinical settings but also community fields without clinical expert requirement.

Study of the occurrence of tropane alkaloids in animal feed using LC-HRMS by Ana Romera-Torres; Roberto Romero-González; José Luis Martínez Vidal; Antonia Garrido Frenich (3340-3346).
The consumption of animal products is rapidly increasing and, therefore, the use of feedstuffs in farms is in demand in developing countries. Feed cereals represent more than 48% of total feed material and they might be contaminated by Solanaceae plants, which contain toxic metabolites such as tropane alkaloids. A wide-scope analysis of tropane alkaloids in feed, including atropine, scopolamine, tropinone, anisodamine, tropane, homatropine, tropine, littorine, physoperuvine, pseudotropine, apoatropine and aposcopolamine, has been performed using liquid chromatography coupled to an Exactive-Orbitrap analyzer. An extraction method based on pressurized liquid extraction with methanol/water/formic acid (75/25/0.4; v/v/v) as a solvent extraction, followed by a solid phase extraction step using Strata-X-C cartridges and a clean-up step with chitosan has been used. The method was fully validated and recoveries from 70 to 109% with an intraday precision lower than or equal to 15% were achieved and limits of quantification (LOQs) ranged from 5 to 25 μg kg−1 for all the compounds. More than 40 samples belonging to several classes of animal feed (cow, rabbit, sheep, chicken and pig) were analysed, showing the occurrence of tropane alkaloids in 18 out of 45 samples, with concentration ranging from 5 (atropine) to 222 μg kg−1 (scopolamine), highlighting the need of analytical methods for the control of TAs in animal feeds.

Boron-doped diamond electrode: a modification-free platform for sensitive square-wave voltammetric determination of indapamide hydrochloride by Carlos Alberto Rossi Salamanca-Neto; Fabiana Ayumi Yoshida; Elen Romão Sartori; Jaqueline Tobias Moraes (3347-3352).
A boron-doped diamond electrode (BDDE) was employed for indapamide hydrochloride (IND) determination. IND presented an irreversible and diffusion-controlled oxidation peak at 0.53 V in 0.01 mol L−1 H2SO4 by cyclic voltammetry. To develop the voltammetric method, the effect of electrochemical pretreatment of BDDE, pH of supporting electrolyte, type of electrolyte and its concentration were evaluated. Under optimized instrumental parameters of square-wave voltammetry, the IND current was linear over the concentration range of 0.099–4.3 μmol L−1, with limit of detection of 56 nmol L−1. The method was successfully applied to commercial tablets, and the obtained results were statistically similar to those obtained by a spectrophotometric method. Additionally, the determination of IND was accomplished in synthetic cerebrospinal fluid and tap water. The method proved to not suffer from matrix interference with excellent recoveries.

A novel magnetic solid-phase extraction (MSPE) coupled with high performance liquid chromatography (HPLC) method was explored for aflatoxin B1 (AFB1) and zearalenone (ZON) simultaneous determination. This method was based on a newly explored extractant of Fe3O4 nanoparticle-filled amino-modified multi-walled carbon nanotubes (Fe3O4-MWCNTs-NH2) composite, which was sensitive to analytes with structure of multi-aromatic rings and abundant hydrogen bond acceptors, based on the extraction mechanism of π–π EDA and hydrogen-bonding interactions. The morphology, structure and magnetic properties of the extractant were characterized by transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) and vibrating sample magnetometry (VSM). Adsorption isotherms and kinetics for two mycotoxins were investigated and proved to be the best fit by Dubinin–Astakhov and pseudo-second-order models. Critical MSPE parameters, including solution pH, extraction time, salt addition and temperature, were investigated and optimized and the proposed analytical method was validated. Subsequently, the method was successfully applied for AFB1 and ZON analysis from wheat flour samples. The proposed method proved to be an excellent green analysis with a score of 89 in analytical Eco-Scale evaluation. It also has good linearities in the range of 1.0–100.0 ng g−1 with linear coefficients (r) above 0.99, desirable recoveries ranging from 88.8% to 96.0%, satisfactory precision with relative standard deviation (RSD) in the range of 2.1–2.8%, and low limits of detection (LODs) of 0.15 and 0.24 ng g−1 for AFB1 and ZON, respectively. This novel magnetic-extractant-based green MSPE-HPLC method has great potential for mycotoxin analysis from complex matrix samples.

Use of 3D printing and modular microfluidics to integrate cell culture, injections and electrochemical analysis by Akash S. Munshi; Chengpeng Chen; Alexandra D. Townsend; R. Scott Martin (3364-3374).
Fabrication of microchip-based devices using 3D printing technology offers a unique platform to create separate modules that can be put together when desired for analysis. A 3D printed module approach offers various advantages such as file sharing and the ability to easily replace, customize, and modify the individual modules. Here, we describe the use of a modular approach to electrochemically detect the ATP-mediated release of nitric oxide (NO) from endothelial cells. Nitric oxide plays a significant role in the vasodilation process; however, detection of NO is challenging due to its short half-life. To enable this analysis, we use three distinct 3D printed modules: cell culture, sample injection and detection modules. The detection module follows a pillar-based wall-jet electrode design, where the analyte impinges normal to the electrode surface, offering enhanced sensitivity for the analyte. To further enhance the sensitivity and selectivity for NO detection the working electrode (100 μm gold) is modified by the addition of a 27 μm gold pillar and platinum-black coated with Nafion. The use of the pillar electrode leads to three-dimensional structure protruding into the channel enhancing the sensitivity by 12.4 times in comparison to the flat electrode (resulting LOD for NO = 210 nM). The next module, the 3D printed sample injection module, follows a simple 4-port injection rotor design made of two separate components that when assembled can introduce a specific volume of analyte. This module not only serves as a cheaper alternative to the commercially available 4-port injection valves, but also demonstrates the ability of volume customization and reduced dead-volume issues with the use of capillary-free connections. Comparison between the 3D printed and a commercial 4-port injection valve showed similar sensitivities and reproducibility for NO analysis. Lastly, the cell culture module contains electrospun polystyrene fibers with immobilized endothelial cells, resulting in 3D scaffold for cell culture. With the incorporation of all 3 modules, we can make reproducible ATP injections (via the 3-D printed sample injection module) that can stimulate NO release from endothelial cells cultured on a fibrous insert in the cell culture module which can then be quantitated by the pillar WJE module (0.19 ± 0.03 nM per cell, n = 27, 3 inserts analyzed each day, on 9 different days). The modular approach demonstrates the facile creation of custom and modifiable fluidic components that can be assembled as needed.

Piperazine-tuned NBD-based colorimetric and fluorescent turn-off probes for hydrogen sulfide by Zhong-yong Xu; Zi-ying Wu; Hui-ya Tan; Jin-wu Yan; Xiao-lin Liu; Jia-ying Li; Zi-yan Xu; Chang-zhi Dong; Lei Zhang (3375-3379).
The piperazine ring is a very important factor in governing molecular structure to generate the special properties of a compound. New NBD-based fluorescent probes 1–3 were designed and synthesized for hydrogen sulfide (H2S) detection. Probe 1 showed high sensitivity and selectivity for H2S with a color change from yellow to pink for naked eye observation. However, the analogues (2 and 3) were response-negative toward H2S. In order to clarify the importance of the piperazine ring in the NBD-based H2S probe and the mechanism, the crystal structure of 1 was obtained and the piperazine ring was deemed to mediate H2S nucleophilic addition. In addition, probe 1 had limits of detection of 18.96 μM (UV) and 23.42 μM (FL), and the thiolysis rate for 1 was found to be 0.49 M−1 s−1. Living cell imaging results indicated that probe 1 could be utilized to trace intracellular H2S. This work has a great guiding role in the design of NBD-based fluorescent probes through a nucleophilic addition mechanism.

Electrochemical sensor for the determination of dimetridazole using a 3D Cu2O/ErGO-modified electrode by Xionghui Ma; Jianping Li; Jinhui Luo; Chunhua Liu; Shuhuai Li (3380-3385).
A three-dimensional Cu2O/electrochemically reduced graphene (ErGO)-modified electrode was developed for the determination of dimetridazole. Cu2O/GO was initially prepared using a modified solvothermal method, and the 3D structure was constructed by electrodepositing a Cu2O/GO dispersion on a glassy carbon electrode in the presence of lithium perchlorate. The enhanced electrocatalytic reduction of dimetridazole was confirmed, and the electrochemical reduction reaction was found to be a typical adsorption-controlled process. Our results revealed that the response to dimetridazole was linear in the concentration range of 30–150 nmol L−1 and the detection limit was 3.64 nmol L−1. The prepared electrode was also employed to detect dimetridazole in an egg sample, thereby indicating its potential practical application.

Rapid determination of histamine in fish by thin-layer chromatography-image analysis method using diazotized visualization reagent prepared with p-nitroaniline by Hui Yu; Daina Zhuang; Xingjuan Hu; Shuang Zhang; Zhiyong He; Maomao Zeng; Xubo Fang; Jie Chen; Xiaoe Chen (3386-3392).
A novel analytical method for rapid determination of histamine in fish involves methanol extraction, manual spotting, thin-layer chromatography (TLC) for less than 15 min, visualization by diazotized p-nitroaniline reagent, and quantitation by image analysis using the ImageJ software. Following this procedure, histamine can be successfully detected and completely separated from other biogenic amines and diazonium reagent's positive compounds. Moreover, this method does not require any expensive reagent and instrument as well as any tedious sample pre-treatment. The characterization of this method revealed good specificity, linearity in the range of 100–1200 ng per spot (adjusted R-square = 0.9987), precision (relative standard deviation, 1.07–2.76%), accuracy (with a trueness of 1.76–6.22%), recovery rate (93.78–98.24%), limit of detection (23.49 ng per spot), and limit of quantitation (78.29 ng per spot). The proposed method is simpler, faster and less expensive than HPLC and the previously reported TLC/HPTLC. Therefore, this method has the potential to be adapted by aquatic enterprises and aquatic market supervision commissions for monitoring the quality of seafood products during processing, storage, and sales.

Herein, we present a new and robust adsorbent for the extraction and preconcentration of Hg ions from estuary samples based on the modification of magnesium silicate (MgSi) with 2-mercaptobenzoxazole (MBO). The properties, morphology, and composition of the adsorbent before and after modification were characterized via FT-IR, SEM, and XRD analyses. The concentrated ions were measured using cold vapor atomic absorption spectrometry (CVAAS). The effects of critical parameters such as pH value, eluent conditions, flow rate and volume of the sample were studied and optimized. The calibration curve was linear within the range of 0.3–6.0 ng mL−1 (r = 0.9979). The effect of the major coexisting ions constituting 98% of solutes in natural seawaters was examined using artificial seawater samples. The limit of detection (LOD) of the method was 0.09 ng mL−1 (n = 8) under the optimum conditions. The relative standard deviations (RSD, n = 6) at 0.5 and 4.0 ng mL−1 were 4.3% and 3.9%, respectively. The maximum adsorption capacity of the proposed adsorbent was 58 mg g−1. The developed method was successfully applied to determine ultra-trace amounts of mercury ions in estuary waters with recoveries greater than 97%.

A simple colorimetric analytical assay using gold nanoparticles for specific detection of tetracycline in environmental water samples by Mengyu Qi; Chunyan Tu; Yuanyuan Dai; Weiping Wang; Aijun Wang; Jianrong Chen (3402-3407).
In this work, an aptamer-based colorimetric method for the detection of tetracycline (TC) was established by employing gold nanoparticles (AuNPs) as the colorimetric probe. In the absence of TC, the aptamers are readily adsorbed on the surface of AuNPs, which prevents aggregation of AuNPs and stabilizes the red colloidal solution against high concentrations of NaCl. In the presence of TC, the aptamers are subsequently released from the surface of the AuNPs due to binding with TC molecules. A gradual color change from red to blue was observed due to the salt-induced aggregation of AuNPs. Furthermore, a linear relationship between the absorbance intensity and the concentration of TC was observed in the range of 0.10 to 5.00 μM. The limit of detection was 0.071 μM. The developed assay was employed for the determination of TC in environmental water samples. The recoveries ranged from 106.7% to 115.6% with relative standard deviations being less than 3.0%. The results demonstrate that the method serves as a promising tool for the rapid, simple and cost-effective detection of TC.

An approach combining enzymatic 18O-labeling and label-free methods for the quantitative dynamic analysis of hemogen phosphorylation by Shaohui Sui; Lei Li; Changyan Li; Wanjun Zhang; Weihui Wu; Wenfeng Yu; Zhigang Jiang; Wantao Ying (3408-3415).
Phosphorylation is a key event in many biological processes, and the temporal and quantitative dynamics of protein phosphorylation is crucial to understand the signaling processes in cells. Stable isotope coding is believed to be a powerful technique for relative quantification of the modification level, whereas it is a challenge to obtain the phosphorylation stoichiometry at each phosphosite of the protein. Label-free methods of quantification directly using LC-MS can be introduced to achieve this goal just by comparing the ion peak intensity or spectra counting of a phosphorylated and its corresponding non-phosphorylated peptides eluting in the same run. In this study, we integrated enzymatic 18O-labeling and two kinds of label-free methods to explore the temporal phosphorylation changes over different stimulation times using phorbol myristate acetate (PMA) for hemogen, an important protein in hematopoietic development and neoplasms. Complementary methods lead to the concurrent identification of the relative quantification and the stoichiometry of site-specific phosphorylation. What's more, LC-MS/MS combined with no enzyme specificity searching parameters was applied to assign novel phosphosites.

Multiple sclerosis (MS) is a disease of the central nervous system affecting over 2.5 million people worldwide. MS is characterized by blood brain barrier (BBB) damage and demyelination of axons, which causes brain lesions leading to complications such as numbness and blurred vision. Steroid medications are available to help treat symptoms of MS, and although the mechanism of action of steroids is known for most cells, it is not completely understood with respect to red blood cells (RBCs). Here, we show that the steroids estriol and prednisolone effect RBC ATP release and downstream endothelial NO production, which may be a mechanism of action of steroids in the bloodstream. Our results suggest that while RBCs obtained from people with MS release significantly more ATP than RBCs from healthy people (healthy: 216 ± 11 nM, MS: 321 ± 18 nM), a significant decrease up to 183 nM in ATP release is measured due to the presence of physiological levels of estriol or prednisolone. Further analysis measured a 31% decrease in endothelial nitric oxide (NO) production from endothelial cells in contact with RBCs treated with estriol or prednisolone. These results suggest that steroids may have an additional mechanism of action in the bloodstream through their ability to attenuate RBC ATP release. ATP stimulates NO production in vivo, and while high levels of NO are detrimental to the BBB, it follows that hindering the amount of ATP in the bloodstream would decrease the BBB damage and demyelination in MS.

Back cover (3423-3424).