Analytical Methods (v.5, #13)

Front cover (3155-3155).

Inside front cover (3156-3156).

Contents list (3157-3167).

Electrochemical biosensing platform based on a hemocyanin–Au@QC NP–carbon black hybrid nano-composite film by Peng Yang; Jun You; Fangping Li; Junjie Fei; Bo Feng; Xiulan He; Jinping Zhou (3168-3171).
Novel gold nanoparticles (Au@QC NPs), using quaternized cellulose (QC) as the support matrix, were used to entrap hemocyanin (Hc) with the aid of carbon black. Direct electron transfer between Hc and the electrode was achieved and an electrochemical method for the simultaneous detection of hydroquinone and catechol is developed.

Direct HPILC analysis of cellulose depolymerisation in ionic liquids by Kosuke Kuroda; Yukinobu Fukaya; Hiroyuki Ohno (3172-3176).
Changes of average molecular weight and molecular weight distribution of cellulose in a polar ionic liquid (IL) were analysed with high performance liquid chromatography using a polar IL as an eluent (HPILC). 1-Ethyl-3-methylimidazolium methylphosphonate was used as the polar IL. As a model of partly depolymerised cellulose, a mixed sample composed of cellulose and cello-oligosaccharides (glucose, cellobiose, cellotetraose, and cellohexaose) was evaluated to test the resolution of the HPILC. In the resulting chromatograms, the corresponding peaks for each saccharide were found. Hydrolysed cellulose catalysed by a cellulase mixture in water was then prepared and dried. Then, this was dissolved in the polar IL to analyse its molecular weight distribution. The molecular weight distribution changed depending on the enzymatic reaction time. The peak for cellulose was found to decrease with the increase of the peak for cellobiose, and subsequently the peak for cellobiose decreased with the increase of that for glucose. In addition, cellulose oligomers except for cellobiose were scarcely observed, showing the catalytic feature of cellulase. Depolymerisation of cellulose in the polar IL was also carried out using ultrasonication. The peak for cellulose in the HPILC profiles shifted to a higher retention volume side and broadened with the sonication time, strongly suggesting random depolymerisation of cellulose. Thus, HPILC was confirmed to be effective for the dynamic analysis of cellulose depolymerisation.

A portable, pressure driven, room temperature nucleic acid extraction and storage system for point of care molecular diagnostics by Samantha Byrnes; Andy Fan; Jacob Trueb; Francis Jareczek; Mark Mazzochette; Andre Sharon; Alexis F. Sauer-Budge; Catherine M. Klapperich (3177-3184).
Many new and exciting portable HIV viral load testing technologies are emerging for use in global medicine. While the potential to provide fast, isothermal, and quantitative molecular diagnostic information to clinicians in the field will soon be a reality, many of these technologies lack a robust front end for sample clean up and nucleic acid preparation. Such a technology would enable many different downstream molecular assays. Here, we present a portable system for centrifuge-free room temperature nucleic acid extraction from small volumes of whole blood (70 μL), using only thermally stable reagents compatible with storage and transport in low resource settings. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis of simulated samples demonstrate a lower limit of detection of 1000 copies per mL, with the ability to detect differences in viral load across four orders of magnitude. The system can also be used to store extracted RNA on detachable cartridges for up to one week at ambient temperature, and can be operated using only hand generated air pressure.

An ultra-high-performance liquid chromatography tandem mass spectrometry confirmatory method using pneumatically assisted electrospray ionisation (UPLC-ESI-MS/MS) was developed for the simultaneous quantification of licit and illicit drugs in whole blood across multiple drug classes. The method was validated on 51 frequently detected drugs and metabolites in human ante-mortem and post-mortem whole blood. The generic sample treatment procedure was based on deproteinisation by a mixture of methanol and acetonitrile followed by ultrafiltration of the extract. Matrix-matched calibrants combined with isotope dilution of almost all substances were used for quantitative analysis. A high sample throughput was obtained by using a 96-well format. The total instrumental run-time between injections was 16 min. The relative intra-laboratory reproducibility standard deviations were generally below 8% and 15% at concentrations of 250 μg L−1 and 5 μg L−1, respectively. The mean true recoveries were in most cases greater than 90% for ante-mortem samples and greater than 80% for post-mortem samples. For most substances, the limits of detection and quantification were less than 1 μg L−1 and 3 μg L−1, respectively.

Solid phase extraction (SPE) is an essential component in many proteomic analyses involving matrix-assisted laser desorption/ionization mass spectrometry detection (MALDI-MS). Parametric variables affecting the efficiency of protein extractions from buffer (Tris–HCl) and urine media using a capillary-channeled polymer (C-CP) fiber stationary phase in a tip-based format are evaluated. Proteins are immobilized on to polypropylene (PP) C-CP fibers and eluted in organic solvents conducive to high analytical performance using a simple benchtop centrifuge to drive the fluids. Experimental variables, including elution solvent strength, aqueous wash volume, the interactive aspects of fiber tip length (2.5–10 mm), and loading and elution volumes, were evaluated to gain insights into fundamental processes and to optimize protein recoveries/analytical signals. Based on the MALDI-MS responses of the test proteins cytochrome c, lysozyme, and myoglobin, the optimized elution solvent was determined to be a 60 : 40 ACN–H2O mixture with 0.07% trifluoroacetic acid (TFA). It was determined that different aqueous wash volumes were required to flush test solution remnants, depending on the buffer concentration, concluding that a 100 μL wash was effective at concentrations of up to 1 M. Fiber tip length was explored to determine the limits of loading volume/protein mass for each bed size while efficiently extracting from the buffer environment. Interestingly, the shortest C-CP fiber tips provided the best efficiency, isolating nanogram levels of protein from 1 μL aliquots of sample. After optimization, a proof of practice experiment was performed to extract the three-protein suite (<5 μM of each) from a synthetic urine matrix. The previously undetected proteins could be readily distinguished with high spectral clarity due to the SPE procedure utilizing C-CP fiber packed micropipette tips.

Affinity analysis of somatostatin and somatostatin receptor by surface plasmon resonance by Jinhang Gao; Huan Tong; Zhiyin Huang; Rui Liu; Xian Li; Ou Qiang; Chengwei Tang (3201-3206).
For drug development and quality monitoring targeted at somatostatin (SST), a sensitive and reliable method is needed to detect the binding of somatostatins (SSTs) to their receptors – somatostatin receptors (SSTRs). In this study, interactions between SSTs (SST14, SST28) and SSTRs (SSTR2, SSTR4) have been measured by using surface plasmon resonance (SPR). SSTRs were covalently immobilized on the surfaces of the CM5 sensor chip via amine coupling and SSTs were injected into the flow channels. The results revealed that SST14 bound to SSTR4 with a much higher affinity than to SSTR2. Meanwhile, SST28 bound to SSTR4 with a slightly higher affinity when compared to SSTR2. SST14 and SST28 interacted with SSTR2 with a similar affinity, but SST14 had a relatively higher affinity of interaction with SSTR4 when compared to SST28. Furthermore, our study demonstrated that SSTR2-CM5 is capable of quantitating SST14 and SST28 with a detection range of 5 μg mL−1 to 50 μg mL−1 and 5 μg mL−1 to 40 μg mL−1, respectively. Therefore, the SPR technique not only offers a potentially sensitive and reproducible method for SSTR-selective drug discovery, but also might provide a rapid and quantitative bioassay for monitoring manufactural batch consistency.

A new method for the detection of latent fingermarks on paper surfaces using p-dimethylaminobenzaldehyde (DMAB) is described. The method is based upon the reaction of DMAB with the amino acids present in the latent fingermark to give a yellow-brown impression that is photoluminescent when illuminated with a high intensity filtered light source at 490 nm and viewed through orange goggles (OG550). A wet contact method proved effective on non-fragile porous substrates such as white photocopy paper and various other substrates, while a dry contact (solventless) method afforded development on thermal paper. Luminescence spectrophotometry of developed l-alanine, glycine and l-serine spots on paper was used to confirm that DMAB was reacting with amino acids in the latent fingermark.

Monitoring changes in the chemical composition of dissolvable tobacco products by Christina L. Rainey; Jennifer J. Berry; John V. Goodpaster (3216-3221).
Dissolvable tobacco products are smokeless, spit-free, made from finely milled tobacco, and dissolve in the mouth of a user. In 2009, R.J. Reynolds Tobacco Co. released a line of dissolvable tobacco products in several test markets. In late 2010, the dissolvables were reformulated and several changes in their composition were determined and are reported here. Our analytical approach featured gas chromatography-mass spectrometry preceded by four separate methods for sample preparation: solvent extraction using acetone, trimethylsilyl derivatization of sugars, solid-phase microextraction of semi-volatiles and liquid–liquid extraction for the quantitative analysis of nicotine. Taken together, these methods identified several changes in the formulation of the initial products and their reformulated versions. One major change in the new dissolvables is that “mellow” and “fresh” flavors have been replaced by a “mint” flavor. Additionally, the strips are no longer dominated by glycerol, but rather threitol. Total nicotine and (free nicotine) concentrations for the orbs, sticks, and strips were found to be 3.35 mg g−1 (1.242 mg g−1), 2.6 mg g−1 (0.850 mg g−1), and 2.74 mg g−1 (0.572 mg g−1), respectively. Total nicotine and free nicotine concentrations have significantly increased in some cases.

Pyrene-appended, benzimidazoliums-urea-based ratiometric fluorescent chemosensor for highly selective detecting of H2PO4 by Xiaozhi Jiang; Dawei Zhang; Jinjiang Zhang; Jianwei Zhao; Binshen Wang; Meiyun Feng; Zhiyun Dong; Guohua Gao (3222-3227).
A novel flexible pyrene-based chemosensor 1 bearing two benzimidazolium moieties and a urea group was designed and synthesized. The binding properties of chemosensor 1 toward various anions were examined via UV-vis, fluorescence, and 1H NMR spectroscopic methods. The results demonstrated that chemosensor 1 could distinguish H2PO4 from other tested anions such as F, Cl, Br, I, AcO and HSO4 by ratiometric fluorescence as chemosensor 1 formed a complex with H2PO4 in 2 : 2 ratio via intermolecular excimer transduction in CH3CN. The fluorescence response of chemosensor 1 to H2PO4 was hardly interfered by other anions. The detection limit of chemosensor 1 (5 × 10−6 M) toward H2PO4 is 5.02 × 10−7 M.

A sensitive and simple method for the determination of ellagic acid (EA) was developed based on the fluorescence quenching effect of EA for glutathione (GSH)-capped CdTe quantum dots (QDs). Under optimum conditions, a good linear relationship was obtained from 0.3118 to 55.0 μg mL−1 with a correlation coefficient of 0.9983, and the detection limit (3σ/K) was 0.0936 μg mL−1. The fluorescence quenching mechanism has been proposed on the basis of electron transfer supported by ultraviolet-visible (UV-vis) absorption, fluorescence (FL) spectroscopy and the measurement of fluorescence lifetime. The method has been applied to the determination of EA in synthetic samples and fresh urine samples of healthy humans with satisfactory results. The proposed method manifested several advantages such as high sensitivity, short analysis time, low cost and ease of operation.

A new electrochemical immunoassay strategy with signal amplification was designed for sensitive detection of free prostate-specific antigen (fPSA), using electroactive silver-mediated poly(amidoamine) (PAMAM) dendrimer nanostructures as molecular tags on the polyclonal rabbit anti-human PSA antibody (pAb1)-modified screen-printed carbon electrode (SPCE). The molecular tag was first synthesized and functionalized with monoclonal mouse anti-human prostate-specific antibody (mAb2). In the presence of target fPSA, a specific sandwich-type immunocomplex was formed on the functionalized SPCE, and the carried silver nanoparticles could directly catalyze the reduction of hydrogen peroxide in the detection solution without the participation of bioactive enzymes. Compared with silver nanoparticles alone, the presence of PAMAM dendrimer increased the immobilized amount of silver nanotags and enhanced the detectable signal of the electrochemical immunoassay. Two labeling protocols including silver nanoparticle-labeled mAb2 and silver-PAMAM-labeled mAb2 were implemented for the detection of fPSA, and improved analytical features were achieved with the silver-PAMAM labeling. Under the optimal conditions, the developed immunoassay enabled the determination of target fPSA with a wide dynamic range of 0.005–5.0 ng mL−1 and a detection limit (LOD) of 1.0 pg mL−1 (S/N = 3). The simple preparation and strong attachment of silver-PAMAM nanostructures with pAb2 resulted in a good repeatability and intermediate reproducibility down to 7.5%. In addition, the accuracy of the electrochemical immunoassay was evaluated for the detection of 7 clinical serum specimens, achieved in good accordance with the referenced enzyme-linked immunosorbent assay (ELISA) method.

Here we report a simple colorimetric method for the detection of adenosine triphosphate disodium (ATP) using unmodified gold nanoparticles (AuNPs) as an optical probe. In acidic media, the melamine can link the neighboring AuNPs and result in the formation of the gold nanoparticle aggregates. Simultaneously, the color of AuNPs solution changes from red to blue. However, upon the addition of ATP, the responsive disassembly of the gold nanoparticle aggregates occurs, due to the competitive adsorption between the ATP and the melamine molecules on the surface of the AuNPs. Consequently, the aggregated AuNPs are dispersed, accompanied by the naked-eye visible color change from blue to red. Thus, a reverse assay procedure was established to light up the colorimetric sensing of ATP on basis of the ATP-triggered responsive disassembly of the melamine-crosslinked gold nanoparticle aggregates, and the logarithm of absorbance ratio of A660/A518 was found to be proportional to the ATP concentration over the range of 0.6–2.4 × 10−7 mol L−1 with a detection limit (3σ) of 8.0 nM.

Simultaneous detection of controlled substances in waste water by Ellen M. Mwenesongole; Lata Gautam; Sarah W. Hall; John W. Waterhouse; Michael D. Cole (3248-3254).
This study presents a method of simultaneous detection of both traditional and newly emerged drugs of abuse in wastewater. The method is based on solid phase extraction and gas chromatography-mass spectrometry analysis. This analytical method separates 25 drugs from different classes including amphetamines, cathinones, tropane alkaloids, piperazines plus ketamine, amitriptyline, diazepam and morphine. In addition, newer compounds (methcathinone, mephedrone, butylone), and isomers (1-(2-methoxyphenyl) piperazine, 1-(4-methoxyphenyl) piperazine; 1-(2-flurophenyl) piperazine, 1-(4-flurophenyl) piperazine; 1-(3-trifluoromethylphenyl) piperazine, 1-(4-trifluoromethylphenyl) piperazine) have been separated, with greater sensitivity (×100 order of magnitude). This work reports the detection of butylone, mephedrone, 1-(4-methoxyphenyl) piperazine, 1-(2-flurophenyl) piperazine and 1-methyl-4-benzylpiperazine for the first time in waste water. This suggests that with changes in drug use patterns, constant monitoring of waste water entering treatment plants should be carried out and treatment processes need to be put in place for their removal.

A simple and relatively selective solid phase extraction system has been described and used for determination of trace amounts of Ni(ii) ions in water, soil and split peas by sodium dodecyl sulfate (SDS)-coated microparticles of walnut sawdust modified with dimethylglyoxim. Scanning electron microscopy (SEM) and Fourier transformed infrared (FT-IR) techniques were used for characterization of the sorbent. Adsorption experiments were carried out using batch mode under various operating conditions. The effect of pH, contact time, ionic strength, volume and concentration of the eluent on the Ni(ii) preconcentration were studied. The equilibrium time was found to be 30 min and the adsorbed ions were stripped from the solid phase by 10 mL of 3 mol L−1 nitric acid. The eluting solution was analyzed for the Ni(ii) content by flame atomic absorption spectrometry (FAAS). The adsorption data were measured at room temperature and the yielded Langmuir monolayer capacity was 22.0 mg g−1. The detection limit (LOD) and the relative standard deviation were 0.55 μg L−1 and 3.1% (n = 5, concentration = 0.1 mg L−1), respectively. The accuracy of the method was examined with the determination of nickel ions in a certified reference material (CRM TMDW-500, drinking water).

Evaluation of poly(vinylpyridine)-supported protoporphyrin resin for the sampling/separation of manganese(ii) using a hyphenated FIA-FAAS system by Talita Fogaça de Oliveira; Fernanda Midori de Oliveira; Mariana Gava Segatelli; César Ricardo Teixeira Tarley (3264-3271).
The present study describes the development of a preconcentration method for manganese determination by FAAS using poly(protoporphyrin-co-vinylpyridine) as a sorbent. To synthesize this material, 4-vinylpyridine and protoporphyrin were copolymerized in the presence of ethylene glycol dimethacrylate as a cross-linker and 2,2-azobisisobutyronitrile as an initiator. The factors affecting the flow injection preconcentration system were optimized by employing 25 fractional factorial and Doehlert matrix designs. The proposed on-line preconcentration method was performed by percolating 18.0 mL aliquots of Mn(ii) solutions (pH 8.3) at a flow rate of 6.0 mL min−1 through a mini-column, packed with 100 mg of poly(protoporphyrin-co-vinylpyridine); the Mn(ii) ions were then on-line eluted with 1.5 mol L−1 HNO3 into the FAAS nebulizer. Under these conditions, it was possible to yield a linear curve in the concentration range of 0.0–120.0 μg L−1, with a correlation coefficient of 0.997. Besides, the following system parameters were obtained: sampling frequency of 15 h−1, preconcentration factor of 53, consumptive index of 0.34 mL, concentration efficiency of 17.6 min−1, detection limit of 0.34 μg L−1, and quantification limit of 1.13 μg L−1. The method precision (repeatability) estimated for ten measurements of 1.0 and 50.0 μg L−1 Mn(ii) standard solutions was found to be 5.2 and 4.9%, respectively. Moreover, the protoporphyrin incorporation into the poly(vinylpyridine) network caused a remarkable 323% increase in the method's sensitivity. Finally, the proposed procedure was not affected by potentially interfering ions. Thus, it was successfully applied to the Mn(ii) determination in water, food and sediment samples.

A bare fused silica capillary lacks enough efficiency for electrophoretic separation under some circumstances as a complex mixture of analogous compounds or stroma matrix. To improve the separation, a strategy of functionalization for the inner surface with nano-silica and polyelectrolytes is proposed in this paper. The results have demonstrated that the hybrid coating film of poly(diallyldimethylammonium chloride) (PDDA)–nano-SiO2–poly(styrenesulfonate) (PSS) on a silica capillary greatly promoted the separation efficiency. As a model, four analytes (naringenin, rutin, quercetin and ascorbic acid) are well separated with 24 mmol L−1 borate buffer solution (pH 8.3) as the carrier. The proposed approach was successfully applied for rapid determination of these analytes in artificial urine with satisfactory results.

[(E)-3-(2-chlorophenyl)-1-(2-hydroxy-4,6-dimethoxy-3-(1,2,3,6-tetrahydropyridine-4-yl)phenyl)-2-propylene-1-one], L41, was found to be a potent cyclin-dependent kinase 1 inhibitor. To study its pharmacokinetic characteristics, a simple, specific, sensitive and reproducible HPLC method was developed and validated to quantitatively determine L41 in rat plasma. L41 and amlodipine besylate (internal standard, IS) were extracted from rat plasma by a simple liquid–liquid extraction process with ethyl acetate. Chromatographic separation was performed on an Agela C18 column and an isocratic mobile phase [methanol–water (containing 50 mM ammonium acetate), 77 : 23, v/v, pH 6.5] at a flow rate of 1 mL min−1 with a total run time of 10 min. The UV absorbance at 343 nm was recorded. L41 and IS eluted at 5.1 and 8.9 min, respectively. The calibration plot was linear over the concentration ranging of 15–1200 ng mL−1 (r > 0.998). The intra- and inter-day precisions of analysis were <12% and accuracy ranged from 93.5 to 106.3%. The validated HPLC method was successfully applied to the pharmacokinetic study of L41 in rats after a single intravenous and three oral administrations.

A simple, rapid and selective sample pretreatment method, molecularly imprinted matrix solid-phase dispersion (MI-MSPD) coupled with liquid chromatography, was developed for the rapid isolation of acyclovir (ACV) from edible creatural tissues. New molecularly imprinted polymer (MIP) synthesized by using theophylline as a dummy template revealed a high special affinity to ACV, and was employed as the selective dispersant of matrix solid-phase dispersion (MSPD) for the rapid screening of ACV. The presented method obviously reduced the tedious separation procedure, improved the extraction selectivity and purification efficiency, and significantly eliminated the effect of template leakage of MIP on quantitative analysis. The results indicated that the interference compounds originating from the creatural tissue matrix could be efficiently eliminated, and the elution was clean enough for HPLC analysis. Good linearity of ACV was obtained in a range of 0.10–50.0 μg g−1 with r2 = 0.9998, and the limit of quantification based on the signal to noise of 10 was 0.09 μg g−1. The recoveries at three spiked levels were in the range of 85.5–108.1% with RSD ≤ 3.7% (n = 3). The presented MI-MSPD method combined the advantages of MIP and MSPD, and could be applied for the rapid and selective screening of ACV in creatural tissues.

Uncertainties estimation for determination of 10 elements in Northeastern China black bee honey by ICP-MS by Hui Chen; Chunlin Fan; Zhibin Wang; Qiaoying Chang; Guofang Pang (3291-3298).
A method for the determination of 10 elements, including Na, Mg, P, K, Ca, Mn, Zn, Rb, Sr and Ba, in black bee honey sourced from Heilongjiang Province, Northeastern China is described. Tea (GBW10016) was chosen as the Certified Reference Material (CRM) for method validation. The “bottom-up” approach was used to estimate the sources of uncertainty, such as repeatability, weighing of the sample, recovery, sample volume, sample dilution, stock solution and calibration curve. The range of relative expanded uncertainties for all of the elements was 3.70 to 17.11% (coverage factor k = 2, providing a level of confidence of approximately p = 95%). The percentage contribution of each source to the relative combined uncertainties was calculated. The data indicated that the major contributions to the uncertainty budget originating from the calibration curve, repeatability and recovery. The validated method was successfully applied to determine 10 elements in Northeastern China black bee honey gathered from different sampling sites. This study was a part of the Northeastern China black bee honey traceability project. We believe that the method used in this work can be generally applied to determine other elements in other samples.

Study of controlled degradation processes and electrophoretic behaviour of omeprazole and its main degradation products using diode-array and ESI-IT-MS detection by Juan José Berzas Nevado; Gregorio Castañeda Peñalvo; Rosa M. Rodríguez Dorado; Virginia Rodríguez Robledo (3299-3306).
This paper provides chemical information of controlled degradation processes on omeprazole, and its main degradation products in aqueous solutions, using CE as the analytical technique. Both diode-array and ESI-IT-MS were employed as detectors in order to generate analytical data of the drugs studied. This information could be used to determine potential impurities for pharmaceuticals of omeprazole because it may influence not only the therapeutic efficacy but also the safety of active pharmaceutical ingredients. For this purpose, a new capillary zone electrophoresis method using diode-array detection for the simultaneous determination of omeprazole and its main degradation products was developed. Some different less aggressive degradation processes were studied, the fastest of which was found to be that of omeprazole in aqueous solutions exposed to UV light (254 nm). Using a background electrolyte (10 mM phosphate buffer, pH 12) the analytes were determined in less than 6 min. Electrophoretic behaviour using an optical detector revealed the presence of seven products under the different experimental conditions used. More analytical chemical information was obtained on omeprazole degradation products using direct infusion electrospray ionization-mass spectrometry operating in negative ion mode [M − H], and even some chemical structures of omeprazole degradation products have been proposed.

An on-line separation and preconcentration system coupled with flame atomic absorption spectrometry for the determination of lead by Teslima Daşbaşı; Şerife Saçmacı; Serkan Şahan; Ahmet Ülgen; Şenol Kartal (3307-3313).
A simple on-line separation/preconcentration procedure is described for the determination of trace levels of Pb(ii) by flame atomic absorption spectrometry (FAAS). A mini-column packed with Dowex Marathon C (DMC) ion-exchange resin was used for the on-line preconcentration of Pb(ii) at pH 3.5. Pb(ii) was sorbed on the resin, from which it could be eluted with 3 mol L−1 HCl and then introduced directly to the nebulizer–burner system of FAAS. The parameters influential on the determination of Pb(ii) ions such as the pH of sample solution, eluent type, interfering ions and flow variables were studied. Under the optimum conditions, the calibration graph obtained was linear over the concentration range of 0.01–0.1 mg L−1. The detection limit of the method was 1.3 μg L−1 while precision was 1.1% (n = 15) at a 0.05 mg L−1 Pb(ii) level. The accuracy of the method was proven using standard reference materials. The developed method has been applied successfully to the determination of lead in water and various environmental samples with satisfactory results.

A cyclic voltammogram of aclonifen was obtained on a glassy carbon electrode at pH 4.0 during the potential scan from 0.00 mV to 1400 mV vs. Ag/AgCl (3 mol L−1 NaCl). From repetitive cyclic voltammograms, one anodic peak at +1175 mV and one cathodic peak on the reverse scan at +350 mV were recorded. The sensitive square wave anodic peak at +1220 mV was linearly related to aclonifen concentration ranging from 0.245 to 3.70 μg mL−1. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.073 μg mL−1 and 0.245 μg mL−1, respectively. The percent recoveries of aclonifen calculated from soil and water samples spiked with 1.0 μg mL−1 level were 98.80% and 102.40% with the relative standard deviations of 1.82 and 3.61%, respectively. The method was also applied to the herbicide formulation Challenge® 600 and the average content of 595.3 ± 9.73 g L−1 (n = 7, % 95 confidence interval) was consistent with the 600 g L−1 aclonifen level quoted by the manufacturer. The considerably high recoveries and low standard deviations provide evidence for the high accuracy and precision of the recommended method.

A LC-UV method to assay N-acetylcysteine without derivatization: analyses of pharmaceutical products by Aline Ferreira Ourique; Karine Coradini; Paula dos Santos Chaves; Solange Cristina Garcia; Adriana Raffin Pohlmann; Silvia Stanisçuaski Guterres; Ruy Carlos Ruver Beck (3321-3327).
A reversed-phase liquid chromatography (LC) method with UV detection was developed and validated for the assay of N-acetylcysteine (NAC) in granules and effervescent tablets. The method involves the use of the same chromatographic conditions and avoids any derivatization step. The separation was done using a reversed-phase column LC-18, as the stationary phase, and a mixture of 0.05 M KH2PO4 and acetonitrile (95 : 5 v/v) containing 0.095% (v/v) of phosphoric acid, as the mobile phase. UV detection was performed at 214 nm and a linear response was observed over the concentration range between 10 and 50 μg mL−1 of NAC. The method was revealed to be suitable and was successfully validated in accordance with the official guidelines for specificity, linearity (r = 1), detection and quantification limits (0.70 and 2.11 μg mL−1), precision (RSD < 2%), accuracy (recovery > 97%), and robustness (RSD < 4%). Therefore, NAC can be assayed in granules and effervescent tablets using the same chromatographic conditions without derivatization.

Electrochemical bisphenol A sensor based on carbon nanohorns by Guifang Xu; Lingshan Gong; Hong Dai; Xiuhua Li; Shupei Zhang; Shuangyan Lu; Yanyu Lin; Jinghua Chen; Yuejin Tong; Guonan Chen (3328-3333).
Herein, a new carbon material, carbon nanohorns (CNHs), was firstly employed as the sensing element to fabricate a simple electroanalytical platform for detecting food born contaminants. Bisphenol A (BPA) was chosen as the model to be tested in this sensing platform. The detailed electrochemical behavior of BPA was investigated on the modified electrode and its kinetic parameters were further calculated. The relative results pointed out that CNHs, which have a similar electrocatalysis effect as previously reported carbon materials, like graphene and carbon nanotubes, could obviously amplify the electrochemical oxidation response of BPA because of its unique and charming properties. The experimental conditions affecting the BPA determination were optimized. The relative results indicate the potential application of CNHs in designing sensitive and convenient sensors for the rapid and on-site monitoring of food born contamination.

Setting up the chromatographic analysis of anthelmintics using the “Crossed D-Optimal” experimental design methodology by Nikolaos G. Margaritelis; Catherine K. Markopoulou; John E. Koundourellis (3334-3346).
An “experimental design” methodology was applied to optimize the chromatographic separation and quantitative determination of six anthelmintics levamisole (LEV), thiabendazole (THI), mebendazole (MEB), albendazole (ALB), praziquantel (PRA) and niclosamide (NICL) by HPLC. Based on the “Crossed D-Optimal” criterion, the participation of three mixed solvents in the mobile phase (methanol, water and acetonitrile) was investigated, since binary systems presented low chromatographic resolution. Furthermore, this approach provides an opportunity to use a crossover design to study the effects of other chromatographic parameters, such as pH, which is considered important for achieving optimal results. Sample analysis was carried out on an HS C18 column (250 × 4.6 mm) 5 μm at 40 °C and the detection was performed with both a UV-diode array detector and an ELSD (Evaporative Light Scattering Detector). The mobile phase consists of an aqueous ammonium acetate buffer (pH 5.5; 0.05 M), acetonitrile and methanol (40 : 37 : 23, v/v/v) with 1 mL min−1 flow rate. The system was found to produce sharp and well resolved peaks for all analytes with their retention times ranging from 3.5 to 13.0 min. Linear regression analysis for the calibration curves showed a good relationship with regression coefficients higher than 0.999 for the UV detector and 0.997 for the ELSD respectively. The proposed method was validated and proven to be accurate, precise, reproducible and suitable for routine analyses.

In this article, we propose very simple procedures to analyze two important indole alkaloids, reserpine and yohimbine, in Rauvolfia yunnanensis. A nonaqueous CE method has been employed using berberine as an internal standard. Optimum separation conditions were obtained when the sample was injected by pressure for 3 s at 50 mbar and then separated with the buffer containing 40 mM ammonium acetate, 2.0% (v/v) acetic acid, and 20% (v/v) acetonitrile in methanol medium at an applied voltage of 25 kV. The analytes were detected at 220 nm. The two alkaloids can be separated within 15 min and quantified with high sensitivity. Good recoveries were obtained with both the procedures, using an internal standard. Finally, these procedures were applied to the analysis of two alkaloids in Rauvolfia yunnanensis.

In this paper, we established a micellar electrokinetic chromatography method for fast and simultaneous determination of nitrendipine and atenolol in new antihypertensive combination tablets. Buffer conditions were optimized by using a multivariate response surface methodology (RSM) established by Box–Behnken Design in terms of sodium dodecyl sulfate concentration, buffer concentration and pH of buffer. Under the optimum buffer conditions, the separation of the two drugs can be finished in 3 minutes in a 31.2 cm × 50 μm fused-silica capillary at an applied voltage of 25 kV and the temperature of 25 °C. The optimal running buffer (pH 8.9) was Na2B4O7 (7.5 mmol L−1) and NaH2PO4 (30 mmol L−1) containing 15 mmol L−1 sodium dodecyl sulfate. Good correlation coefficients were found (γ2 >0.999) at concentrations of 5–17.5 μg mL−1 for nitrendipine and 10–35 μg mL−1 for atenolol, respectively. All the RSD results of precision experiments were below 3%. The recoveries of nitrendipine and atenolol were 98.98–100.15% and 99.46–101.18%, respectively. The limits of detection of this method were 1 μg mL−1 and 0.5 μg mL−1 for nitrendipine and atenolol and the limits of quantification of this method were 2.5 μg mL−1 for nitrendipine and 1.5 μg mL−1 for atenolol. After the optimization, the method was successfully applied for the content uniformity test according to the United States Pharmacopeia.

In this paper, a simple and highly sensitive electrochemical sensor based on a macroporous carbon modified glassy carbon electrode (MPC/GCE) was developed and used for detecting luteolin in phosphate buffer solution (PBS, pH 7.0) for the first time. Cyclic voltammetry (CV) and differential pulse voltammetric methods (DPV) were applied to investigate the electrochemical behavior and mechanism of luteolin detection on the MPC/GCE. The results indicated that MPC/GCE can greatly enhance the electrocatalytic activity towards the redox of luteolin. A series of experimental parameters including the pH of the supporting electrolyte, accumulation potential and time were optimized. In neutral solution, the MPC/GCE showed a low detection limit of 1.3 × 10−9 M (S/N = 3), a favorable ability of anti-interference to electroactive molecules and good reproducibility and long-term stability. Finally, the method was successfully applied for the determination of luteolin in a capsule of Lamiophlomis rotata Kudo (Chinese patent medicine) with satisfying results.

Peptide-tight ESI/MSn analysis with segment of liquid chromatography effluent by Lei Nie; Guobin Xu; Ji Ji; Qingping Liu; Huizhi Fan; Baohong Liu; Pengyuan Yang (3371-3378).
A segment flow protocol has been proposed for coupling nano-liquid chromatography (nano-LC) with mass spectrometry (MS) in peptide-based proteomics research. LC effluent was segmented by perfluorodecalin oil and stored as segments in sequence to carry out the following off-line MSn analysis. In this way, incomplete identification of co-eluted peptides resulting from the MS scan rate, relatively slower than the LC elution rate, was solved to some extent for on-line coupling LC-MSn analysis. The complex proteins from a sample of T. tengcongensis were successfully identified with one run using segment as the interface between nano-LC and MSn analysis, compared to the total identified proteins with six runs using routine on-line nano-LC-MSn analysis. This segment flow approach could eliminate detection loss and sample cross-contamination, providing a new method for peptide-based proteomic research, especially when the amount of sample is limited.

A specific sandwich-type electrochemical immunosensor for the sensitive detection of neuron-specific enolase (NSE) is described by using platinum nanoflower (PtNF)-labeled horseradish peroxidase-anti-NSE conjugate (HRP-mAb2) as a secondary antibody on a carbon nanosphere-functionalized graphene sensing platform. In the presence of the target NSE, the sandwiched immunocomplex is formed on the functionalized graphene sensing interface between the labeled mAb2 on the platinum nanoflowers and the immobilized primary antibody (pAb1) on the electrode. Upon addition of hydrogen peroxide to the detection solution, the horseradish peroxidase can catalyze the reduction of the hydrogen peroxide with the aid of the immobilized thionine on the electrode in pH 5.5 acetic acid buffer. Under the optimal conditions, the cathodic current of the as-prepared immunosensor increases with the increasing NSE concentration, and exhibits a dynamic linear range from 0.01 to 120 ng mL−1 with a detection limit of 5.0 pg mL−1 NSE at S/N = 3. Intra- and inter-assay coefficients of variation were below 10%. The methodology was evaluated by assaying spiked serum samples, and the variation coefficients were 1.9–3.6%.

An ultra high performance liquid chromatography electrospray ionization mass spectrometry method was developed for the analysis of a broad range of boronic acids present in industrial samples. A set of 7 boronic acids having different functional groups was successfully separated on an Acquity BEH C18 column using 10 mM ammonium acetate and acetonitrile as the mobile phase. The proposed method has excellent potential for high-throughput analysis as it enjoys the advantage of one minute run time without using any sample pretreatment or pre-derivatization which has been mandatorily followed during boronic acid analysis to avoid dehydration. The influence of specific instrument parameters of UPLC-MS was examined and an optimized condition has been proposed for the identification of boronic acids and to reduce the predominant formation of boroxine, solvent adduct and dimer ions which normally complicate the analysis of boronic acids. The proposed method was further validated by analyzing a wide range of 20 boronic acids having different functional groups. The estimated recovery values of the boronic acids were ranged from 97.1 to 105.7%, and precision, expressed as relative standard deviation, was below 2.0% and the linearity R2 was 0.98 based on UV response. This method provides LOD of 0.1 μg and LOQ of 1.0 μg for the majority of boronic acids and was successfully applied for the quantitative analysis of boronic acids in industrial applications such as reaction monitoring samples from Suzuki coupling, arylation of amines and phenols.

Choline-like ionic liquid-based aqueous two-phase extraction of selected proteins by Songyun Huang; Yuzhi Wang; Yigang Zhou; Li Li; Qun Zeng; Xueqin Ding (3395-3402).
In this paper, an ionic liquid-based aqueous two-phase system (ATPs) has been developed for the effective extraction of bovine serum albumin (BSA). Compared with the effects of three different ionic liquids used in an aqueous two-phase system, a choline-like ionic liquid was selected as the suitable solution for bovine serum albumin. Extraction efficiencies of BSA were found to be dependant on the temperature, time, IL mass and the salt content. Based on single-factor experiments, these significant operating parameters of the ILs-ATP were respectively optimized. Experimental results show that 92.03–100.03% of the proteins could be extracted into the ionic liquid-rich phase in a single-step extraction. Repeatability, precision and stability experiments were investigated and the RSDs are lower than 2.3%, 1.7% and 4.7% respectively. Experiments indicated that intermolecular forces, the micelle phenomenon and the salting-out effect play important roles in the transfer process, and the aggregation phenomenon was the main driving force for the separation. Conformation of the protein was not affected after extraction into the upper ionic liquid-rich phase according to the determination by UV-visible (UV-vis), fluorescence spectra, dynamic light scattering (DLS), Fourier transform infrared (FT-IR) spectroscopy and TEM. It is suggested that the method of choline-like ILs-based ATPs for the selective separation of protein would have potential applications in bio-analysis and bio-separation.

Evaluation of calibration approaches for quantification of pesticide residues in surface water by SPE with small-size cartridges followed by fast GC-MS by Andrea Purdešová; Svetlana Hrouzková; Mária Andraščíková; Zoltán Krascsenits; Eva Matisová (3403-3409).
A fast GC-MS method for analysis of twenty four GC amenable pesticides in surface water was developed and validated. Extraction of pesticides using solid phase extraction (SPE) with small-size cartridges was performed. Four calibration approaches for quantification purposes, (1) acetonitrile (MeCN) standards, (2) MeCN standards with analyte protectants (APs), (3) matrix-matched standards, and (4) matrix-matched standards with APs, were investigated and statistically compared in terms of validation parameters. Linearity was studied in the concentration range of 0.01–2.5 μg L−1 with coefficients of determination (R2) higher than 0.9466. Satisfactory recoveries at three studied concentration levels (0.05, 0.1 and 1 μg L−1) in the range of 65.6–100.3% with relative standard deviation <19% were obtained. The limits of detection (LODs) and limits of quantification (LOQs) were evaluated for all approaches. LODs ranged from 0.06 to 20 ng L−1 and LOQs from 0.2 to 65 ng L−1. The developed and validated method was applied for the analysis of Danube River water samples.

Polyfluorinated alkyl substances (PFASs) are among emerging persistent organic pollutants (POPs) that are widely present in a variety of consumer products. The typical approach for determination of volatile PFASs in ambient air is based upon the high-volume active sampling and solvent extraction (method A), which suffers from the poor portability, high noise level and significant loss of low molecular weight analytes. All these limitations have restricted its application in urban regions, especially in residential areas when conducting source apportionment analysis. In this study, method A was compared with another approach using low-volume air sampling and thermal desorption (method B). In general, method B showed a better repeatability and recovery, while method A demonstrated a lower detection limit. Application of both methods in the outdoor urban environment of Singapore indicated a higher time resolution, higher portability and lower noise level of method B. With these advantages method B appeared to be more suitable for the quantitative analysis of targeted PFASs in regions with concentrated point and variable diffusive sources.

A new method for the determination of trans-resveratrol in grape seed was developed by coupling liquid extraction (LE) and micellar electrokinetic chromatography (MEKC). LE was employed for sample preparation and MEKC for analytical separation. The solvent used for LE was ethyl acetate and the buffer for MEKC consisted of 25 mM borate containing 25 mM sodium dodecyl sulfate (SDS) and 2.5 mM sodium salt sulfated-β-cyclodextrin at pH 9.0. The limit of detection (S/N = 3) was found to be 4.62 × 10−7 g ml−1. Linear regression of peak area versus concentration of resveratrol was checked with the r squared being greater than 0.99 for resveratrol at a concentration range of 9.25 × 10−6 g ml−1 to 4.63 × 10−2 g ml−1. The recoveries were over 90% for the determination of resveratrol in grape seed samples. The method developed can be an attractive alternative to the HPLC methods reported in the literature for the determination of resveratrol in grape seed with the advantages of a reduction of analysis time and operation cost. To the best of our knowledge, this is the first report on the determination of resveratrol in grape seed based on coupling LE and MEKC.

Back cover (3423-3424).