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Analytical Methods (v.4, #6)

Front cover (pp. 1461-1462).

This study aims to develop a protocol for classifying sex and age groups of deceased persons by using the chemical information in burnt bone fragments. Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) has been employed to assess the chemical compositions of the burnt bones from the deceased, all of Thai nationality. By performing three ICP-OES measurements on burnt bone samples from 75 individuals, 43 males and 32 females, with an accurate record of their age at death, only six elements i.e. Al, Ca, K, Mg, P, S, were observed in the fragments above the instrumental detection limit of ICP-OES. Fifteen concentration ratios of these particular elements were examined for their significant difference with respect to sex and age group by Mann–Whitney U and Kruskal–Wallis tests, respectively. Subsequently, the significantly different ratios were employed to assess the relationship between bone chemical compositions and sex/age groups of the deceased using logistic regression analysis. Binary and multinomial logistic regressions were required to classify sex and age group at death, respectively. Regression analyses indicated that the input variables representing sex should be included for classifying the age group of the deceased. The chemical information in burnt bone fragments could be employed to assess the sex and age groups of the deceased with 79.60% and 75.10% accuracy, respectively. Chemical measurements were performed on burnt bone samples from two additional individuals to verify the developed logistic equations and classification protocol. More fractions of bones from an individual give a higher percentage of correction for classifying sex and age at death of the deceased.

Contents list (pp. 1463-1481).

Matrix solid-phase dispersion (MSPD) and modified QuEChERS methods were compared for the extraction of pesticide residues from onion followed by determination by liquid chromatography with electrospray ionization tandem mass spectrometry. The efficiency of the methods was statistically compared using recovery and precision data, matrix effects and other extraction characteristics such as the sample mass, solvent volume, extraction time and limits of detection and quantification (LOQ). In general, faster extractions and lower LOQ values (0.0005 to 0.05 mg kg⁻¹) were achieved with QuEChERS, whereas greater ruggedness and lower matrix effects were obtained with MSPD. For both methods, high extraction yields were achieved (61.8–120%) with relative standard deviations lower than 20%. Matrix effects were observed for both methods, and were compensated by using matrix-matched calibration.

Quality control of routine sampling in chemical analysis by Analytical Methods Committee, AMCTB No 51 (pp. 1482-1483).

In sampling terminology, the ‘target’ is the mass of material that the customer needs to characterise, usually to support a decision about its acceptability or price. For most such materials there is an established sampling protocol regarded as fit for purpose. This is mainly because all targets are actually or potentially heterogeneous. But even when a protocol is followed exactly, repeat samples differ in composition from each other and from the target. Moreover, there is a distinct possibility that the target may be atypically heterogeneous. These circumstances, particularly the last, mean that a sample may not be fit for purpose, even when the sampling is executed strictly according to the protocol. And, of course, there is always the possibility that the sample was not collected according to the protocol! As either of these conditions might give rise to a suboptimal decision, it would be helpful to know when such an event occurred. That would call for some form of quality control on the sampling process.

Pharmaceutical Analysis by Melissa Hanna-Brown (pp. 1484-1484).

We have developed a simple and sensitive method for the simultaneous determination of the antiviral drug oseltamivir (OS) and its metabolite oseltamivir carboxylate (OSC) in environmental waters by automated online in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). OS and OSC were separated within 4 min using an ODS-3 column and 1% acetic acid (aq)/methanol (55/45, v/v) as the mobile phase at a flow rate of 0.2 mL min⁻¹. Electrospray ionization conditions in the positive ion mode were optimized for MS/MS detection of OS and OSC. The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 μL of sample at a flow rate of 200 μL min⁻¹ using a CP-Pora PLOT amine capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC-MS/MS method, we obtained good linearity of the calibration curve (r

Study on the interaction behavior of catalase with cephalosporins by chemiluminescence with flow injection analysis by Donghua Chen; Zhuming Wang; Yun Zhang; Xunyu Xiong; Zhenghua Song (pp. 1485-1487).

The interaction behavior of catalase with cephalosporins was first studied using luminol as luminescence probe. By chemiluminescence model of protein–drug interaction in flow injection system, the binding parameters of catalase with cephalosporins were obtained, giving the binding ability of cephalosporins to catalase and the thermodynamic parameters of catalase/cephalosporins association process.

Detection of native chondroitin sulfate impurities in heparin sodium with a colorimetric micro-plate based assay by Timothy K. Toby; Cynthia D. Sommers; David A. Keire (pp. 1488-1491).

We have recently described a 96-well plate format assay for visually detecting oversulfated chondroitin sulfate A (OSCS) in heparin that uses a water soluble cationic polythiophene polymer (3-(2-(N-(N′-methylimidazole))ethoxy)-4-methylthiophene (LPTP)) and heparinase digestion. Because dermatan sulfate (DS, a.k.a. chondroitin sulfate B (CSB)) and, to a lesser degree, chondroitin sulfate A (CSA) are the most common native impurities of heparin active pharmaceutical ingredients (APIs) we adapted the previously established micro-plate assay for the visual detection of DS and CSA in heparin sodium. We describe a naked-eye detection test of these common heparin impurities with sensitivity appropriate for the United States Pharmacopeia (USP) 1% percent galactosamine in total hexosamine specification. For example, the test detects DS ≥ 1.0% w/w visually and 0.5% using a plate reader, or CSA ≥ 1.0 to 2.0% w/w visually and at ≥1.0% with a plate reader. In contrast to the test developed for oversulfated glycosaminoglycans such as OSCS, the LPTP–chondroitinase test developed here utilizes chondroitinase ABC instead of heparinases and requires a centrifugal filtration step to remove un-digested heparin. The test takes advantage of the sensitivity of the LPTP chemosensor to low molecular weight glycosaminoglycan (GAG) fragments formed by digesting DS or CSA. Importantly, the LPTP–chondroitinase method detects DS and CSA in heparin sodium in a format potentially amenable to high throughput screening.

Enantioanalysis of ketoprofen based on its interaction with C60 fullerene and its derivatives by Raluca-Ioana Stefan-van Staden; Rahel Girmai Bokretsion (pp. 1492-1497).

Three enantioselective, potentiometric membrane electrodes (EPMEs) based on C60 fullerene and its derivatives, (1,2-methanofullerene C60)-61-carboxylic acid and tert-butyl(1,2-methanofullerene C60)-61-carboxylate, were designed for the enantioanalysis of ketoprofen. Molecular modelling has been used to investigate the enantioselective binding between the enantiomers of ketoprofen and C60 fullerenes and to prove the mechanism of potential development for the proposed electrodes. The slopes obtained for these electrodes are near-Nernstain with detection limits of 10⁻⁸ and 10⁻⁷ mol L⁻¹ magnitude order. The proposed electrodes can be reliably used for the enantioanalysis of ketoprofen raw material as well as from its pharmaceutical formulations. Electronic structures as well as molecular interaction have been investigated using Hartree–Fock theory, 3-21G(*) basis set. Stability and feasibility of all the generated structures were supported by their respective energy minima and fundamental frequencies. Molecular modeling calculations were in good agreement with the performances of the proposed electrodes.

NMR reaction monitoring during the development of an active pharmaceutical ingredient by Ian M. Clegg; Charles M. Gordon; David S. Smith; Roberto Alzaga; Anna Codina (pp. 1498-1506).

We show the use of NMR spectroscopy to develop detailed understanding of a reaction mechanism during development of a manufacturing process for an active pharmaceutical ingredient (API). This includes structural elucidation of reaction starting materials, intermediates and products. The method is able to generate an immediate mass balance through quantitative profiling of all materials involved in the reaction. A straightforward reaction monitoring methodology was employed using existing laboratory based equipment.

A new and simple PLS calibration method for NIR spectroscopy. API determination in intact solid formulations by Marcelo Blanco; Anna Peguero (pp. 1507-1512).

The NIR spectra of pharmaceuticals are the result of component contribution and the effects of the different steps of the production process (granulation, compaction, and coating). These effects, even though they are of low magnitude, affect significantly the results of calibration models. Incorporating such effects into the calibration set is essential with a view to constructing a model capable of accurately predicting the contents of production tablets. We developed a new method for incorporating the variability introduced by the production process in the spectra for the calibration set. The method calculates the difference between the spectrum of a tablet and a powder mixture of identical composition prepared in the laboratory; the differences thus calculated for several tablets constitute a set of mathematical vectors that define the overall variability matrix of the process: the process variability matrix. This matrix is added to a set of NIR spectra for several powder mixtures (prepared in laboratory) spanning the desired content range for the active pharmaceutical ingredient (API) in order to obtain the spectral matrix for the calibration set. The API content (in %) of calibration samples is established from weights of their components in the laboratory powder mixtures. The calibration model is constructed by applying the partial least-squares (PLS) algorithm to the spectral matrix of the calibration set. This methodology has been applied successfully on API determination in commercial pharmaceutical tablets.

Automated analysis of oseltamivir and oseltamivir carboxylate in environmental waters by online in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry by Hiroyuki Kataoka; Haruna Shiba; Keita Saito (pp. 1513-1518).

Determination of anticonvulsants in human plasma using SPME in a heated interface coupled online to liquid chromatography (SPME-LC) by Claudete Alves; Paulo C. F. de Lima Gomes; Álvaro José dos Santos-Neto; José Carlos Rodrigues; Fernando Mauro Lanças (pp. 1519-1524).

A simple and sensitive method using solid phase microextraction (SPME) and liquid chromatography (LC) with heated online desorption (SPME-LC) was developed and validated to analyze anticonvulsants (AEDs) in human plasma samples. A heated lab-made interface chamber was used in the desorption procedure, which allowed the transference of the whole extracted sample. The SPME conditions were optimized by applying an experimental design. Important factors are discussed such as fiber coating types, pH, extraction time and desorption conditions. The drugs were analyzed by LC, using a C18 column (150 mm × 4.6 mm × 5 μm); and 50 mmol L⁻¹, pH = 5.50 ammonium acetate buffer : acetonitrile : methanol (55 : 22 : 23 v/v) as the mobile phase with a flow rate of 0.8 mL min⁻¹. The suggested method presented precision (intra-assay and inter-assay), linearity and limit of quantification (LOQ) all adequate for the therapeutic drug monitoring (TDM) of AEDs in plasma.

Chromatographic determination of active compounds in topical formulations by Ludmila Matysová; Petra Koblová; Lubomír Galla; Hana Sklenářová; Lucie Havlíková; Petr Solich (pp. 1525-1529).

The aim of this study was to develop and validate new methods for the determination of active compounds in topical gels. The method for the determination of nonoxinol-9 and trimecaine hydrochloride in lubricant gel by HPLC with UV detection was described. Separation was carried out using a Discovery ZR-PBD analytical column (150 × 4.6 mm, 5 μm), and a mobile phase composed of a mixture of triethylammonium acetate buffer (pH 9; 50 mM) and methanol (22 : 78, v/v). Determination of terpinen-4-ol in lubricant gel was achieved by GC with a flame ionization detector (FID). An Alltech-AT-624 (0.32 mm ID × 30 m, 1.8 μm film thickness) fused-silica capillary column and helium as the carrier gas, at a flow rate of 28 cm s⁻¹, were employed. The analysis time was less than 7.5 min for HPLC and 5 min for GC analyses. Both methods were successfully applied to the analysis of pharmaceuticals.

Compression effect on sustained-release and water absorption properties of cellulose tablets studied by heterospectral two-dimensional (2D) correlation analysis by Hideyuki Shinzawa; Kimie Awa; Yukihiro Ozaki (pp. 1530-1537).

Chemical and physical effects of the compression process on cellulose excipients are explored by near infrared (NIR) spectroscopy, X-ray diffraction (XRD), dissolution test and water absorption test. A set of pharmaceutical tablets including pentoxifylline (PTX) and cellulose are prepared under varying levels of compression. The compressive deformation of the tablets is probed by NIR spectroscopy and XRD. The interaction of tablet with water molecules is also analyzed with the dissolution and water absorption tests. The essential relationship between the two different classes of data, e.g. XRD and dissolution (or absorption) test, is effectively elucidated by heterospectral two-dimensional (2D) correlation analysis. It revealed that the compression produces a disordered amorphous component of cellulose. Such development of the mobile amorphous phase results in a more tightly packed matrix with less porosity. Thus, in turn, it prevents penetration of water molecules into the tablet and direct contact with PTX, which eventually brings the delay in the dissolution. Consequently, by carrying out the hetero-correlation analysis of the two datasets, it effectively provided a more detailed picture of the compression process.

Automated analysis of non-steroidal anti-inflammatory drugs in human plasma and water samples by in-tube solid-phase microextraction coupled to liquid chromatography-mass spectrometry based on a poly(4-vinylpyridine-co-ethylene dimethacrylate) monolith by Qiong-Wei Yu; Xin Wang; Qiao Ma; Bi-Feng Yuan; Hai-Bo He; Yu-Qi Feng (pp. 1538-1545).

A weak anion exchange polymer monolith based on poly(4-vinylpyridine-co-ethylene dimethacrylate) (poly(VP-co-EDMA)) was prepared in the capillary. The use of polyethylene glycol (PEG) as porogens and EDMA as crosslinking monomers helps to increase the specific surface area and enhance hydrophobicity of the target monolith. The monolith exhibited satisfactory permeability, high mechanical strength and good stability in aqueous buffer. The potential application of the monolith as extraction medium in complicated samples has been demonstrated by in-tube solid-phase microextraction (in-tube SPME) of three non-steroidal anti-inflammatory drugs (NSAIDs) (ketoprofen, fenbufen and ibuprofen) in human plasma and environmental water followed by on-line liquid chromatography-mass spectrometry (LC-MS) analysis. Integration of the sample extraction, LC separation and MS detection into a single system allowed for direct analysis of the NSAIDs in diluted plasma or environmental water sample, which facilitates the realization of automation of the sampling, extraction, separation and detection within a short period of time. The results showed that the limits of quantitation (S/N = 10) for the three NSAIDs were 6.70–15.9 ng mL⁻¹ in human plasma and 0.65–1.87 ng mL⁻¹ in water from East Lake. And the recoveries of the three NSAIDs spiked in human plasma and water from East Lake were from 88.17% to 112.48%, with relative standard deviations less than 13.87%. Compared to previously reported off-line sample extraction and conventional LC methods, the newly developed automated in-tube SPME/HPLC-MS in the current study provided a powerful tool for the fast and sensitive analysis of three NSAIDs in human plasma and environmental water.

An assay of DNA by resonance light scattering technique and its application in screening anticancer drugs by Xi Chen; Guoliang Liu; Shengwang Liang; Sihua Qian; Jinbin Liu; Zhanguang Chen (pp. 1546-1551).

In this contribution, a novel assay has been developed to detect the interaction between DNA and drugs using ethidium bromide (EB) as a probe based on the decreased resonance light scattering (RLS) technique. It was found that, in pH 2.0 Britton–Robinson buffer solution, EB underwent dramatic enhancement of RLS signal when binding to DNA. Additionally, the strong RLS signal of the EB–DNA system was remarkably decreased after the addition of DNA-targeted anticancer drugs. The results showed a linear relationship between the enhanced RLS intensity of the EB–DNA system and concentration of DNA in the range of 0.015–40.8 μg mL⁻¹. The linear regression equation is represented as follows: ΔIRLS = 4.15 + 21.14c with regression coefficient r = 0.9970 (n = 10). The detection limit was 0.3 ng mL⁻¹. Only 2.0 × 10⁻⁸ mol L⁻¹ was selected as the optimum EB concentration in this assay. Further study demonstrated that the anticancer efficacy of anticancer drugs varied inversely with RLS intensity of the EB–DNA-drug system. So, it is intuitive to see the sequence of antitumor efficiency of anticancer drugs without data processing by RLS screening spectra. Compared with the traditional cell-based screening methods, the proposed method was more convenient, rapid and intuitive. The reaction mechanism is discussed.

Determination of volatile residual solvents in pharmaceutical products by static and dynamic headspace liquid-phase microextraction combined with gas chromatography-flame ionization detection by Mohammad Saraji; Taghi Khayamian; Zahra Hassanzadeh Siahpoosh; Bahman Farajmand (pp. 1552-1559).

Headspace single-drop microextraction (HS-SDME) and dynamic headspace liquid-phase microextraction (DHS-LPME) combined with GC-FID were used to determine thirteen residual solvents in pharmaceuticals. Different extraction conditions such as microdrop volume, ionic strength of the sample, extraction time, stirring rate, extraction temperature, solvent volume, sampling volume and number of extraction cycles were studied. n-Octanol was used as the extracting solvent for both methods. Under the optimal conditions, the limits of detection for the analytes obtained by DHS-LPME and HS-SDME ranged from 1.9 to 44 μg L⁻¹ and 0.3 to 21 μg L⁻¹, respectively. Enrichment factors ranged 9–212 and 15–379 for DHS-LPME and HS-SDME, respectively. The relative standard deviations (RSDs, n = 5) for both methods are lower than 9.1%. The applicability of the selected method (HS-SDME) in real sample analysis was investigated.

Development and validation of a new and rapid HPLC for determination of lyophilized teicoplanin by Mariana Henrique Passoni; Hérida Regina Nunes Salgado (pp. 1560-1564).

A sensitive, precise and specific high performance liquid chromatographic method was developed for the assay of teicoplanin in injectable pharmaceutical form. Analytical parameters were studied according to the International Conference on Harmonization (ICH). The method validation parameters yielded good results and included the range, linearity, precision, accuracy, specificity and recovery. The HPLC separation was carried out by reversed phase chromatography on a Waters symmetry C18 column (250 × 4.6 mm id, 5 μm particle size) with a phase composed of acetonitrile : methanol (50 : 50, v/v), pumped isocratically at a flow rate of 1.0 mL min⁻¹. The effluent was monitored at 279 nm. The developed HPLC method to determine lyophilized teicoplanin can be used to evaluate the quality of regular production samples.

A simple UV-photolysis digestion method for the determination of fluoride in fluorine-containing drugs by ion-selective electrode and spectrophotometry techniques by M. V. Balarama Krishna; S. V. Rao; V. S. N. Murthy; D. Karunasagar (pp. 1565-1572).

A simple, effective and reliable UV photolysis digestion (UVPD) method has been developed for the determination of fluoride in pharmaceuticals containing fluorine as one of the constituents. It is based on the use of high intensity UV-irradiation in the presence of a mixture of HNO3 and methanol. For the optimization of the UVPD procedure, three bulk drug samples namely levofloxacin, nebivolol and efavirenz, were chosen as representatives of three diverse matrices: containing single fluorine, two fluorine atoms and trifluoromethyl groups respectively. Optimization of the procedure involves an initial reaction with nitric acid followed by a UVPD approach for complete mineralization of the sample i.e., conversion of bound fluorine to free fluoride ion followed by determination by fluoride ion-selective electrode (F-ISE) and spectrophotometry based methods. Parameters affecting the UVPD method, such as acid concentration, digestion time and sample weight, were optimized to obtain the quantitative recovery (>95%) of fluoride. Quantitative recovery of the fluorine in the samples could be achieved within 20–30 min of UV exposure time for a 50-mg sample. Total digestion of the drug samples was carried out by a closed microwave digestion system and the recoveries of the fluoride were found to be low (<70%) demonstrating the capabilities of the UVPD procedure. The proposed UVPD method was successfully applied to various commercially available fluorine containing drug samples.

A sensitive chemiluminescence determination of isoproterenol in pharmaceutical and human serum using luminol–diperiodatoargentate(iii) system by Behzad Rezaei; Ali A. Ensafi; Fariba Haghighatnia (pp. 1573-1578).

A novel injection analysis-direct chemiluminescence (CL) method has been developed for rapid, sensitive and simple determination of isoproterenol (ISP) in pharmaceutical and human serum samples. The method is based on the enhancement effect of ISP on CL signal of the reaction system, luminol–diperiodatoargentate(iii) in alkaline solution. Under optimum conditions, the CL intensity of luminol–diperiodatoargentate(iii) is proportional to ISP concentration in the range of 1.0 × 10⁻⁸ to 9.0 × 10⁻⁷ mol L⁻¹. The detection limit was found as 4.7 × 10⁻⁹ mol L⁻¹ ISP, and the relative standard deviation for eleven replicated measurements of 2.0 × 10⁻⁷ mol L⁻¹ ISP was 2.7%. The reaction mechanism for the CL reaction is proposed and discussed.

Application of a glassy carbon electrode modified with functionalized multi-walled carbon nanotubes as a sensor device for simultaneous determination of acetaminophen and tyramine by Jahan Bakhsh Raoof; Reza Ojani; Mehdi Baghayeri; Mohaddeseh Amiri-Aref (pp. 1579-1587).

Multi-walled carbon nanotubes (MWCNTs) were oxidatively functionalized in a mixture of sulfuric acid and nitric acid (3 : 1 v/v) while being ultrasonicated. A simple sensitive electrochemical sensor has been developed for selective determination of tyramine (Tyr) in presence of acetaminophen (Ac) by casting functionalized multi-walled carbon nanotubes (fMWCNT) on the surface of a glassy carbon electrode (GCE). The electrocatalytic oxidation of Ac and Tyr is both individually and simultaneously investigated at the surface of the fMWCNT modified glassy carbon electrode (fMWCNT-MGCE) through using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The electrode is able to completely resolve the voltammetric response of Ac and Tyr from potentially interfering species, e.g. ascorbic acid (AA) and dopamine (DA). By use of DPV, linear calibration curves were obtained for Ac and Tyr over the concentration range up to 100 μM and 85 μM in 0.1 M buffered solutions (pH 7.0), respectively. The proposed sensor has limits of detection of 0.42 μM (3σ) for Ac and 0.8 μM (3σ) for Tyr. Finally, it has been demonstrated that the proposed modified electrode can be successfully used for measuring tyramine and acetaminophen in real samples.

Development and validation of a rapid HPLC method for the determination of ascorbic acid, phenylephrine, paracetamol and caffeine using a monolithic column by Petra Koblová; Hana Sklenářová; Ivana Brabcová; Petr Solich (pp. 1588-1591).

This article reports a fast and simple liquid chromatographic method for determination of ascorbic acid, phenylephrine, paracetamol and caffeine. Salicylic acid was used as internal standard. The analytes were successfully separated in less than 5 min by isocratic elution using monolithic column, Onyx Monolithic C18 (100 × 4.6 mm), with mobile phase composed of acetonitrile and phosphate buffer (pH 6.50) (10 : 90, v/v) at a flow rate of 1.0 mL min⁻¹ and 25 °C, sample volume was 10 μL. Detection was observed at two wavelengths 210 nm (phenylephrine, paracetamol and salicylic acid) and 235 nm (ascorbic acid and caffeine). The optimized method was applied for the determination of the analytes in pharmaceutical formulation Coldrex tablets (Smithkline Beecham Consumer Healthcare, United Kingdom) commonly used in virosis treatment.

Optimisation of an HPLC method for the simultaneous determination of pyrantel pamoate, praziquantel, fenbendazole, oxfendazole and butylhydroxyanisole using a phenyl stationary phase by Lucie Havlíková; Ivana Brabcová; Dalibor Šatínský; Ludmila Matysová; Alena Luskačová; Zdeněk Osička; Petr Solich (pp. 1592-1597).

A novel and simple isocratic reversed phase HPLC method with UV and fluorimetric detection for the simultaneous determination of the active anthelmintic components pyrantel pamoate, praziquantel, fenbendazole, its degradation product oxfendazole, and the antioxidant butylhydroxyanisole (BHA) has been developed and validated. The chromatography was performed using a Phenomenex Luna 3 μm phenyl–hexyl column (150 × 3.0 mm), and the mobile phase was composed of 0.5% triethylamine at pH 9.0 and acetonitrile 55 : 45 (v/v) at a flow rate of 1.0 ml min⁻¹. The UV detection was performed at 290 nm for pyrantel, oxfendazole and fenbendazole and at 220 nm for praziquantel. For BHA, fluorescence detection was used with excitation and emission wavelengths of 287 nm and 322 nm, respectively. The method was validated and was applied for the determination of active compounds in various dosage forms of veterinary formulations.

Precision in chemical analysis: a critical survey of uses and abuses by Michael Thompson (pp. 1598-1611).

Precision is a key quantity in assessing the quality of chemical measurement results. It enters into considerations of uncertainty, fitness for purpose, method validation, instrumental performance, internal quality control, proficiency testing, and higher-level activities. The standard deviation of measurement results derived from a single analytical ‘system’ (a combination of a particular analytical procedure and a specific type of test material) depends on many factors, including the conditions of measurement, the state of the test material, and the concentration of the analyte. It is essential that these factors are properly matched to the use to which the precision information will be put.

Identification of enzyme inhibitors using therapeutic target protein–magnetic nanoparticle conjugates by Lin-Sen Qing; Nan Tang; Ying Xue; Jian Liang; Yi-Ming Liu; Xun Liao (pp. 1612-1615).

Target protein–magnetic nanoparticle (MNP) conjugates, i.e.

Sulfite recognition and sensing using Au nanoparticles as colorimetric probe: a judicious combination between anionic binding sites and plasmonic nanoparticles by Jia Zhang; Yue Yuan; Xiaolei Wang; Xiurong Yang (pp. 1616-1618).

A colorimetric sulfite sensor endowed with high selectivity has been developed, using melamine as the anionic binding site and gold nanoparticles as the signaling reporter.

A sensitive and selective label-free DNAzyme-based sensor for lead ions by using a conjugated polymer by Xia Chen; Hongliang Guan; Zhike He; Xiaodong Zhou; Jiming Hu (pp. 1619-1622).

Novel functional oligonucleotides, especially DNAzymes with RNA-cleavage activity, evoke current research interest increasingly due to their potential applications in therapeutics and sensors. One of the most attention-attracting is the lead-specific DNAzyme, which is composed of an enzyme strand 17E and a substrate strand 17S, addition of Pb²⁺ enables the DNAzyme to cleave its substrate. In this study, we took advantage of the unique optical properties of a water-soluble cationic polythiophene (PT) and designed a fluorometric sensing assay for the detection of Pb²⁺. A simple “mix-and-detect” approach enabled the detection of Pb²⁺ within 20 minutes due to the distinguishable optical properties of PT–dsDNA and PT–ssDNA. As low as 10 nM Pb²⁺ could be detected with a detection range from 10 nM to 100 μM via this method. Furthermore, this method was highly selective and only minimally perturbed by nonspecific metal ions. Since common steps such as modification and separation could be successfully avoided, this simple, sensitive, specific, and cost-effective approach showed great potential applications in environmental monitoring, waste management and clinical toxicology.

Effect of oxidative treatment of carbon black on electrochemical activity of cytochrome c by Masayoshi Matsui; Jun-ichi Ozaki (pp. 1623-1625).

Electrochemical activity of cytochrome c (cyt c) adsorbed on as received carbon black (CB) and oxidised CB (oxi-CB) was compared quantitatively using batch adsorption method and cyclic voltammetry. Oxidative treatment of CB was found to be effective in maintaining and promoting the activity of cyt c.

Simultaneous determination of hydroquinone, catechol, resorcinol and nitrite using gold nanoparticles loaded on poly-3-amino-5-mercapto-1,2,4-triazole-MWNTs film modified electrode by Cun Wang; Ruo Yuan; Yaqin Chai; Fangxin Hu (pp. 1626-1628).

A simple and highly sensitive electrochemical method has been developed for the simultaneous determination of hydroquinone (HQ), catechol (CC), resorcinol (RC) and nitrite (NO2⁻). The electrode in this study exhibited high selectivity, well stability and reproducibility and good compatibility.

Multiplex relative quantitation of peptides and proteins using amine-reactive metal element chelated tags coupled with mass spectrometry by Xin Wang; Yangjun Zhang; Xueying Wang; Hongjun Lin; Weijie Qin; Xiaohong Qian (pp. 1629-1632).

Here we describe a chemical labeling method for multiplex relative quantitation of peptides and proteins by coupling metal element chelated tags (MECT) to peptides followed by mass spectrometric analysis.

Bi-detection system for separation-free simultaneous determination of erythrosine and sucrose in candy floss by Xueyan Miao; Wei Wang; Zhen Fang; Bo Xiong; Zitong Wu; Xiaodong Zhou; Jiming Hu (pp. 1633-1636).

A bi-detection system integrated by laser-induced fluorescence (LIF) and retro-reflected beam interference (RBI) based refractive index was established and applied to simultaneously determine erythrosine and sucrose in candy floss. The system realized separation-free analysis of candy floss without prior concentration steps.

Advantages of CE-SDS over SDS-PAGE in mAb purity analysis by Ying Shi; Zhen Li; Jun Lin (pp. 1637-1642).

Traditional SDS-PAGE and a newly developed CE-based method, i.e., CE-SDS, are both widely used to determine mAb purity. This work aimed to compare the two methods side-by-side in details and to establish the pros and cons of each. The sample preparation conditions were first optimized to acquire fair comparison results. When analyzing stressed mAb samples, both methods consistently showed comparable band/peak patterns. The results also suggest both methods are stability indicating. Beyond that, CE-SDS had improved advantages over SDS-PAGE in assay precision, linearity and separation resolution etc. Analysis of the same sample 21 times by utilizing SDS-PAGE introduced large standard deviation in LC and HC percentage determination. Furthermore, SDS-PAGE performed poorly in the linearity testing. The correlation coefficients from the regression were all less than 0.93 (SDS-PAGE) in contrast to those of greater than 0.97 (CE-SDS). When used to separate the non-glycosylated heavy chain (NGHC) from the heavy chain (HC), CE-SDS clearly showed much better resolution over SDS-PAGE. The CE-SDS method was further validated to assess its assay specificity, precision and robustness. For LC, HC and NGHC, the R.S.D. for repeatability, intraday and intermediate precision were determined to be <0.5% for migration time, <2.0% for corrected area percentage and <5.0% for corrected area, respectively. Finally, a small deviation in the migration time and corrected peak areas demonstrated the robustness of the CE-SDS assay when using different capillaries, analysts and sample storage conditions. All these results indicated that the newly developed CE-SDS method is superior to SDS-PAGE, and is suitable for use in areas of biochemical and biological research.

Biomagnetic glass beads for protein separation and detection based on surface-enhanced Raman scattering by Lei Chen; Xiao Xia Han; Zhinan Guo; Xu Wang; Weidong Ruan; Wei Song; Bing Zhao; Yukihiro Ozaki (pp. 1643-1647).

A novel surface-enhanced Raman scattering (SERS)-based detection method for proteins attached to magnetic glass beads have been developed in this study. Silica coated magnetic nanoparticles (Fe3O4) are prepared and aldehyde-functionalized for capturing proteins in solutions by Schiff reaction, which is more biocompatible than previous magnetic nanoparticle and SERS-based detection methods. Target protein and molecule with a specially recognized feature are captured and separated by the magnetic nanoparticles, followed by SERS-based detection. Moreover, these biomagnetic glass beads and detection protocol are found to be useful in protein immunoassay and protein-ligand recognition even in a mixture system (two protein ligands). The proposed magnetic nanoparticles are biocompatible, manipulatable by an external magnetic force and highly sensitive after silver nanoparticles staining, which makes the protocol quite promising for high throughput protein assays.

Statistical analysis of discrete encapsulation of nanomaterials in colloidal capsules by Tatsuya Sakakura; Kazuya Nishimura; Hiroaki Suzuki; Tetsuya Yomo (pp. 1648-1655).

Encapsulation of materials into capsules becomes discrete at small scales. We propose a measurement and analysis method of discrete encapsulation of nanomaterials in colloidal capsules, with the volume of fL to pL, using the fluorescence flow cytometry. Encapsulation of nano-sized polymer beads and lambda-phage DNA into lipid vesicles (liposomes) is evaluated as a model system. By using fluorescence markers, vesicle volume, membrane quantity and nanomaterials in a large number of individual vesicles are measured simultaneously. We show that the number of encapsulated material (k = 0, 1, 2, …) can be evaluated from the quantized fluorescence intensity distribution, which is then compared with the Poisson statistics. As a result, probability of encapsulating beads and λDNA exhibits characteristic trends that depend on the vesicle volume, lamellarity, and electrostatic interaction, which should reflect the physical interaction in the vesicle formation processes. This measurement and analysis will be a powerful tool in designing the colloidal capsules used in many scientific and industrial applications.

Non-invasive monitoring of temperature stress in Arabidopsis thaliana roots, using ion amperometry by Y. Sulaiman; Marc R. Knight; R. Kataky (pp. 1656-1661).

The extracellular storage of calcium of plant cells is one of the two most important calcium stores for signaling and recognized in importance. There has been, for decades, a need in the field of plant calcium signaling to be able to measure the changes in the concentration and dynamics of extracellular calcium in plants, during signaling. We present a novel approach to this problem, and describe the application of ion-amperometry which affords temporal resolution that previous methods could not deliver.

Graphene oxide as a nano-platform for ATP detection based on aptamer chemistry by Wen Dan Pu; Li Zhang; Cheng Zhi Huang (pp. 1662-1666).

Because of its rich π electrons, graphene oxide (GO) can bind with single-stranded DNA (ssDNA) through π-π stacking, but it cannot bind with double-stranded DNA (dsDNA). This different binding ability of GO with DNA has found many applications. By combining this ability with aptamer chemistry, we have developed a highly selective method for the detection of adenosine triphosphate (ATP). The hybrid of ATP aptamer with fluorescein (FAM)-labelled complementary DNA (FAM-DNA) demonstrates a weak affinity for GO and strong fluorescence of FAM-DNA. If ATP is present the strong fluorescence of FAM-DNA is quenched, because the binding of ATP with its aptamer greatly inhibits hybridization of the aptamer with FAM-DNA, and the unhybridized FAM-DNA is adsorbed on to the surface of GO. The quenched fluorescence intensity was found to be in proportion to the concentration of ATP in the range 3–320 μM with a detection limit of 0.45 μM. This method of ATP detection is highly selective and the existence of GTP, CTP and UTP have scarcely effect the determination.

A quantitative assessment of the chemical variation in food grade polyethylene cling film, a common wrapping material for illicit drugs, using attenuated total reflection-Fourier transform infrared spectroscopy by Stephen W. Holman; Trevor F. Emmett; Michael D. Cole (pp. 1667-1673).

Cling film is commonly encountered in forensic investigations as a wrapping for drugs of abuse. The evidential value of a match between a known and a questioned piece of cling film is directly related to the level of the chemical variation both within and between samples. However, little is known about the chemical variation in cling film. To address this, attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) was used to quantitatively assess the chemical variation in food grade low-density polyethylene (LDPE) cling film. Following acquisition of full scan IR spectra between 4000 and 650 cm⁻¹, a spectral region of potential discrimination between 1560 and 1200 cm⁻¹ was identified. The complex series of weak, overlapping absorption bands in this region were deconvoluted and peaks fitted. Bivariate plots comparing each pair of normalised peak areas showed significant overlap of the data points for the different samples, indicating no between-sample chemical variation. Further, the same plots demonstrated that the extent of within-sample chemical variation was at least as great as that between samples. Thus, in the sample set evaluated in this study, it was not possible to discriminate between food grade LDPE cling films using ATR-FTIR spectroscopy due to a lack of quantifiable chemical variation. The study therefore highlights the possibility of false positive matches in the analysis of LDPE cling films and provides a methodology to evaluate the extent of chemical variation in a relevant reference population prior to undertaking casework.

Green extraction using catanionic surfactants of trimethyltetradecyl ammonium bromide–sodium dodecyl sulfate for preconcentration of organophosphorus pesticides in fruit samples by Ketsarin Seebunrueng; Yanawath Santaladchaiyakit; Supalax Srijaranai (pp. 1674-1680).

An extraction method using mixed catanionic surfactants of trimethyltetradecyl ammonium bromide (TTAB) and sodium dodecyl sulfate (SDS) was developed for preconcentration of organophosphorus pesticides (OPPs) before analysis by high performance liquid chromatography (HPLC). The studied OPPs are azinphos-methyl, parathion-methyl, fenitrothion, diazinon, chlorpyrifos, and prothiophos. The optimum extraction condition was 10 mmol L⁻¹ SDS and 0.5 mmol L⁻¹ TTAB in the presence of 10% (w/v) NaCl and equilibration at room temperature. The concentrated target pesticides were subsequently analyzed using a Symmetry C8 column, gradient elution of acetonitrile and water, at a flow rate of 0.8 mL min⁻¹, and detection at 210 nm. Enrichment factors were found in the range of 16–30. The limits of detection of the studied OPPs were 0.001–0.01 mg L⁻¹ which are below the maximum residue limits (MRLs) established by the European Union. Good repeatability (n = 5) and reproducibility (n = 15) were obtained with the relative standard deviation (RSD) below 2% for retention time and below 9% for peak area, respectively. The validation of the proposed method for fruit sample analyses was carried out by matrix matched calibration. A modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method was used for sample preparation before extraction and preconcentration. Recovery of the fortified samples at three levels (0.25, 1.50 and 2.50 mg kg⁻¹) was higher than 80.9% (on average) for all studied fruit samples (pomelo and pineapple). No detectable residues of OPPs in samples were found.

Automatic miniaturized flow methodology with in-line solid-phase extraction for quinine determination in biological samples by A. Margarida Vaz; André R. T. S. Araújo; João L. M. Santos; José L. F. C. Lima; M. Lúcia M. F. S. Saraiva (pp. 1681-1686).

The present work describes an analytical platform based on a multipumping flow injection analysis (MPFS) technique combined with in-line solid-phase extraction (SPE). The flow network has been tested with the determination of quinine in biological samples using fluorometry as the detection technique. Amberlite XAD-4 resin has been used as a solid phase and the implementation of a pH control strategy resulted in a simple and environmental approach for the preconcentration of quinine. Two solenoid valves allowed the deviation of the flow towards the resin column to carry out SPE procedures. The influence of parameters such as concentration, flow rate and volume of the different solutions on the sensitivity and performance of the MPFS was studied. Dynamic calibration ranges (0.78–150 ng mL⁻¹) for quinine determination were applied by using a variable sample volume (120–1000 μL). The developed methodology provided high relative extraction recoveries from human urine samples (85–115%). The proposed automatic methodology turns out to be very efficient and sustainable compared to the available procedures and it could prove to be an attractive alternative tool to perform in-line sample pre-treatment and subsequent direct determination of relevant organic compounds in pharmaceutical and clinical analyses.

Electrochemical detection of dopamine in the presence of epinephrine, uric acid and ascorbic acid using a graphene-modified electrode by Xinying Ma; Mingyong Chao; Zhaoxia Wang (pp. 1687-1692).

A sensitive and selective electrochemical method for the determination of dopamine (DA) in the presence of epinephrine (EP), uric acid (UA) and ascorbic acid (AA) was developed using a graphene-modified electrode. The electrochemical behaviour of DA at the modified electrode was studied by cyclic voltammetry. The results showed that the modified electrode exhibited excellent electrocatalytic activity towards the electrochemical oxidation of DA. When the reduction peak current of DA, in pH 7.0 phosphate buffer solution, was measured at a potential range of −0.1 to 0.6 V, no interference occurred from EP, UA and AA. The reduction peak current was linearly proportional to DA concentration from 2.50 × 10⁻⁶ to 1.00 × 10⁻⁴ mol L⁻¹. The values of correlation coefficient and limit of detection for DA were 0.9984 and 5.00 × 10⁻⁷ mol L⁻¹, respectively. The modified electrode shows excellent sensitivity, selectivity and stability for the voltammetric determination of DA in the presence of EP, UA and AA and was satisfactorily used for the determination of DA in pharmaceutical and biological samples.

Improving the accuracy and precision of an arsenic field test kit: increased reaction time and digital image analysis by James Kearns; Julian Tyson (pp. 1693-1698).

Two procedures to improve the performance of the Hach EZ test kit for quantifying inorganic arsenic concentrations in drinking water have been investigated. In the first, a digital image of the colored spot formed on the test strip, obtained with a flat-bed scanner was analyzed, by the computer program Colors, for the R, G, and B values. Calibrations were constructed by plotting the B values as a function of concentration. Agreement between the experimentally determined B-values and those of the printed chart was only obtained by either increasing the reaction time (to 40 min) or increasing the reaction temperature. The precision as a function of concentration was quantified. A comparison with previously estimated values for visual comparison of the colours, showed that the improved precision of the digital analysis would produce fewer false positive and fewer false negative results at the important threshold values of 10 and 50 μg L⁻¹. By running the test for 24 h, improved performance at the low concentration (around 10 μg L⁻¹) end of the response scale was obtained.

Covalent labeling of mitochondria with a photostable fluorescent thiol-reactive rhodamine-based probe by Shuqi Wu; Yunlong Song; Zhu Li; Zhisheng Wu; Jiahuai Han; Shoufa Han (pp. 1699-1703).

Fluorescent imaging of mitochondria is an essential tool for studies of mitochondrial functions. The staining of mitochondria with potential-indicating dyes, e.g., rhodamine 123, readily vanishes upon loss of the transmembrane potential under certain conditions. 1-(Rhodamine B)-4-(2′-chloroacetyl)-piperazine amide (RB-CAP) was shown to be electrophoretically accumulated into mitochondria, forming covalent bioconjugates with intramitochondrial protein sulfhydryls which enabled the mitochondrial staining to endure in subsequent collapse of the transmembrane potentials. RB-CAP is highly photostable and exhibits stringent selectivity in covalent labeling of mitochondria in living cells. Being much less expensive, RB-CAP is a superior substituent for MitoTracker probes in functional studies of mitochondria.

Feature extraction for identification of drug body packing based on nonnegative matrix factorization by Wei Li; Dingjun Qu; Minqiang Li; Jinyun Liu; Yu Zhong; Fang Zhang; Bai Sun; Daoyang Yu; Jinhuai Liu (pp. 1704-1708).

In the analysis of energy dispersive X-ray diffraction (EDXRD) spectra of drug body packing, feature extraction is a great challenge. In this work, nonnegative matrix factorization (NMF) is proposed to identify drug body packing. NMF was applied to extract features from EDXRD spectra of samples in a set of drugs and other materials concealed in an anthropomorphic phantom. Compared with the features extracted by principal component analysis (PCA) and robust PCA, the features extracted by NMF are physically significant, and can be easily interpreted as diffraction peaks of samples. The features were classified by K-nearest neighbor and support vector machine. The results indicated that the recognition rate using NMF was ideal (above 98%) and insensitive to classifiers. This investigation has demonstrated that NMF is effective in feature extraction for the identification of drug body packing.

Detection and removal of mercury and lead ions by using gold nanoparticle-based gel membrane by Yen-Fei Lee; Fan-Hua Nan; Min-Jane Chen; Hao-Yang Wu; Chih-Wei Ho; Yi-You Chen; Chih-Ching Huang (pp. 1709-1717).

This paper describes the use of two types of bovine serum albumin (BSA)-capped 14.2 nm diameter gold nanoparticles (Au NPs) for the separate detection of mercury (Hg²⁺) and lead (Pb²⁺) ions in highly saline media. The BSA-capped Au NPs were stable in solutions containing up to 500 mM NaCl. Introduction of BSA to a solution of rhodamine 6G (R6G) and 3-mercaptopropionic acid (MPA)-modified Au NPs (R6G/MPA–Au NPs) provided a R6G/MPA–Au NP@BSA probe for the sensing of Hg²⁺ ions. We also used BSA-capped Au NPs to detect Pb²⁺ ions through a mechanism based on Pb²⁺ ions accelerating the leaching rate of Au NPs in the presence of thiosulfate (S2O3²⁻) and 2-mercaptoethanol (2-ME). The resulting deposition of Hg²⁺ ions onto the Au NPs induced the release of R6G from the surfaces of the Au NPs, causing increased fluorescence from the R6G/MPA–Au NP@BSA solution. The Pb²⁺ ions accelerated the dissolution of the 2-ME/S2O3²⁻–Au NPs@BSA into solution, leading to dramatic decreases in the absorption. These two Au NP-based probes were highly sensitive (LOD ≈ nM) and selective (over 100-fold against other metal ions) toward Hg²⁺ or Pb²⁺ ions. We validated the practicality of these two probes through analyses of seawater and urine samples. We also developed a simple gel-based membrane for removal and sensing of Hg²⁺ or Pb²⁺ in aqueous solutions. The agarose gel was used to trap BSA-Au NPs, leading to the preparation of a nanocomposite film of Au NPs@BSA-decorated agarose gel membrane (Au NPs@BSA/AGM) for removing Hg²⁺ or Pb²⁺ in solution. In addition, R6G/MPA–Au NP@BSA-trapped agarose gel membrane (R6G/MPA–Au NP@BSA/AGM) and 2-ME/S2O3²⁻–Au NPs@BSA/AGM allowed for the rapid and simple detection of Hg²⁺ and Pb²⁺, respectively.

Immobilization of chromogenic reagent for the determination of trace cobalt by lab-on-valve bead injection spectroscopy by Yong-Liang Yu; Ying Jiang; Shuai Dou; Jian-Hua Wang (pp. 1718-1724).

An approach for the immobilization of a chromogenic reagent on solid phase micro-beads is performed via a sulfonation reaction. The product as an optical sensor is applied in lab-on-valve bead injection spectroscopy. The benzene ring structure exists in various chromogenic reagents, which facilitates the introduction of a sulfonic acid group via sulfonation. Chromogenic reagents ranging from neutral to anionic can be immobilized on anion exchanger micro-beads. As a model of application, micro-beads of Sephadex QAE A-25 loaded with the chromogenic reagent 1-(2-pyridylazo)-2-naphthol-sulfonic acid (PAN-S) are employed for the determination of trace cobalt by lab-on-valve bead injection spectroscopy. The characteristics of the micro-beads and some important parameters governing the performance of the method are investigated. With a sample volume of 1.0 mL, a detection limit of 8 μg L⁻¹ and a linear range of 20–500 μg L⁻¹ are obtained for cobalt, along with a RSD value of 2.8% (at the 200 μg L⁻¹ level). The accuracy and practical applicability of the present method are validated by analysing a certified reference material of soil GBW07404, vitamin B12 injection and mecobalamin tablets, and further demonstrated by spiking recovery of cobalt in two water samples.

Graphene–Au nanoparticles nanocomposite film for selective electrochemical determination of dopamine by Juan Li; Juan Yang; Zhanjun Yang; Yongfang Li; Suhua Yu; Qin Xu; Xiaoya Hu (pp. 1725-1728).

A novel graphene–Au nanoparticles (AuNPs) composite film modified glassy carbon electrode (GCE) was proposed for selective detection of dopamine (DA). The resulting electrode was characterized using transmission electron microscopy, cyclic voltammetry and differential pulse voltammetry. Compared to bare and graphene modified electrodes, this nanocomposite modified electrode not only significantly improved the electrochemical peak potential difference between DA and ascorbic acid (AA), but also noticeably enhanced the current response. The constructed DA sensor showed a wide linear range (5–1000 μM) and a low detection limit (1.86 μM). The graphene–AuNPs composite, which was obtained by assembling AuNPs onto the graphene surface, could provide a promising platform to develop excellent electrochemical sensors for detecting DA.

A highly selective and sensitive pyridylazo-2-naphthol-poly(acrylic acid) functionalized electrospun nanofiber fluorescence “turn-off” chemosensory system for Ni²⁺ by Sheriff Adewuyi; Dezzline A. Ondigo; Ruphino Zugle; Zenixole Tshentu; Tebello Nyokong; Nelson Torto (pp. 1729-1735).

A fluorescent nanofiber probe for the determination of Ni²⁺ was developed via the electrospinning of a covalently functionalized pyridylazo-2-naphthol-poly(acrylic acid) polymer. Fluorescent nanofibers with diameters in the range 230–800 nm were produced with uniformly dispersed fluorophores. The excitation and emission fluorescence were at wavelengths 479 and 557 nm respectively, thereby exhibiting a good Stokes' shift. This Ni²⁺ probe that employs fluorescence quenching in a solid receptor–fluorophore system exhibited a good correlation between the fluorescence intensity and nickel concentration up to 1.0 μg mL⁻¹ based on the Stern–Volmer mechanism. The probe achieved a detection limit (3δ/S) of 0.07 ng mL⁻¹ and a precision, calculated as a relative standard deviation (RSD) of <4% (n = 8). The concentration of Ni²⁺ in a certified reference material (SEP-3) was found to be 0.8986 μg mL⁻¹, which is significantly comparable with the certified value of 0.8980 μg mL⁻¹. The accuracy of the determinations, expressed as a relative error between the certified and the observed values of certified reference groundwater was ≤0.1%. The versatility of the nanofiber probe was demonstrated by affording simple, rapid and selective detection of Ni²⁺ in the presence of other competing metal ions by direct analysis, without employing any further sample handling steps.

Electrochemical behavior of antipyrine at a Bi2S3 modified glassy carbon electrode and its determination in pharmaceutical formulations by Xiaomeng Meng; Zhenning Xu; Mo Wang; Huanshun Yin; Shiyun Ai (pp. 1736-1741).

A simple electrochemical method based on a Bi2S3 modified glassy carbon electrode (GCE) was developed to determine antipyrine using cyclic voltammetry and differential pulse voltammetry. Antipyrine shows a well-defined oxidation peak at the fabricated electrode in phosphate buffer solution and the oxidation peak current is much higher than that at the bare GCE, indicating that Bi2S3 can effectively improve the oxidation of antipyrine. Several effect factors on antipyrine determination were optimized, such as Bi2S3 amount, solution pH, scan rate and accumulation time. Under the optimal conditions, the oxidation peak current of antipyrine was proportional to its concentration in the range of 2.0 to 100 μM and 100 to 800 μM with a correlation coefficient of 0.9974 and 0.9956, respectively. The limit of detection was estimated to be 0.7 μM (S/N = 3). The developed method showed good reproducibility and excellent anti-interference performance. The fabricated electrode was successfully used to determine antipyrine in pharmaceutical formulations with recovery from 96% to 103.5%.

Feasibility research on rapid detection of dimethoate in water by near-infrared spectroscopy by Yilong Su; Bingren Xiang; Jianping Xu (pp. 1742-1746).

Near infrared (NIR) spectroscopy combined with chemometrics is used for the rapid detection of dimethoate in water without sample preparation. In order to overcome the non-linear phenomenon least-squared support vector machines (LS-SVM) is utilized. Classification accuracy of the training set and the testing set were 100% when two parameters, γ and σ² (270.191 and 29.60, respectively), were chosen by the Simplex optimizing algorithm. The entire operating procedure, which takes less than 2 minutes per sample to complete, is rapid, convenient and environmentally acceptable. It shows potential for the rapid screening of dimethoate in water samples.

Silver nanoparticles in the presence of Ca²⁺ as a selective and sensitive probe for the colorimetric detection of cysteine by Salahaddin Hajizadeh; Khalil Farhadi; Mehrdad Forough; Rahim Molaei (pp. 1747-1752).

A sensitive and selective cysteine colorimeric sensor based on the interaction between cysteine, silver nanoparticles (AgNPs) and calcium ions has been developed. In the presence of Ca²⁺ and NaCl, cysteine could rapidly induce the aggregation of AgNPs, thereby resulting in a yellow-to-red color change. The presence of 10 mM NaCl in the samples decreases the electrostatic repulsion and accelerates the aggregation of AgNPs. Results demonstrated that other amino acids cannot change the color of AgNPs solution in the same conditions, probably due to the absence of the thiol groups, suggesting the selectivity of the proposed method toward cysteine. The color change was monitored by naked eye and UV-Vis spectrophotometry. The ratio of absorption at 524 to 396 nm (A524/A396) is linearly dependent on the cysteine concentration in the range of 0.25–10 μM.

Novel sensor fabrication for the determination of nanomolar concentrations of Ce³⁺ in aqueous solutions by Hasan Bagheri; Abbas Afkhami; Mohammad Saber-Tehrani; Ali Shirzadmehr; Seyed Waqif Husain; Hosein Khoshsafar; Masoumeh Tabatabaee (pp. 1753-1758).

A new Ce³⁺ carbon paste electrode based on a nanocomposite containing multi-walled carbon nanotubes (MWCNTs), nanosilica, room temperature ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate), and 4-(4-methylbenzylideneamino)-5-methyl-2H-1,2,4-triazole-3(4H)-thione (L) as an efficient ionophore was prepared. This sensor responds to cerium ions in a wide linear dynamic range of 2.5 × 10⁻⁸ to 1.0 × 10⁻¹ mol L⁻¹ with Nernstian slope of 19.32 ± 0.10 mV per decade. The detection limit of 7.0 × 10⁻⁹ mol L⁻¹ was obtained at pH range of 3.0 to 8.0. It has a fast response with response time of about 5 s, and can be used for at least 11 weeks without any considerable divergence in the potentials. The proposed sensor displays an excellent selectivity for Ce³⁺ ions with respect to a large number of alkali, alkaline earth, transition and heavy metal ions. This sensor was successfully applied in the determination of cerium ions in aqueous samples.

Keeping particles brilliant – simple methods for the determination of the dye content of fluorophore-loaded polymeric particles by Eva-Maria Laux; Thomas Behnke; Katrin Hoffmann; Ute Resch-Genger (pp. 1759-1768).

One of the most active research areas in the life and material sciences is the design and synthesis of fluorescent nano- and micrometre sized particles for applications e.g. as labels, sensor systems, and platforms for fluorescence assays or barcoding materials. The reliable and reproducible fabrication of such particles as well as many applications require accurate, simple, and versatile procedures for the determination of the dye content per particle which affects e.g. the brightness of these materials and their surface charge and thus, colloidal stability. Here, four fast and inexpensive spectroscopic methods for the quantification of the fluorophore content of beads are presented and compared for nanometre- and micrometre sized polystyrene particles loaded or labeled with commercial fluorophores, differing in dye class, charge, and hydrophilicity. This included the determination of the amount of incorporated dye from absorption spectra of bead suspensions, via dissolving of the polymer matrix, via extraction of the polymer matrix, and from the supernatant of the swelling solution or reaction mixture. Method validation was performed with a sulfur-containing dye and elemental analysis. Based upon this method comparison and the accomplishable uncertainties, two reliable strategies for particle characterization and bead process control are identified that can be easily extended to other materials.

Elemental analysis of burnt human bone for classifying sex and age at death by logistic regression by Kanit Sawasdee; Montip Tiensuwan; Atitaya Siripinyanond; Thamrong Chirachariyavej; Siwaporn Meejoo Smith (pp. 1769-1775).

Electrochemical study of weak inclusion complex interactions by simultaneous MCR-ALS analyses of potential step-chronoamperometric data matrices by Bahram Hemmateenejad; Afsaneh Safavi; Fatemeh Honarasa (pp. 1776-1782).

In this article, the analytical information obtained from the chronoamperometric data of a weak host–guest complexation was analyzed by multivariate curve resolution-alternative least square (MCR-ALS) as a powerful chemometrics method. As a model system, the complexation of dopamine with β-cyclodextrin was investigated. Whilst the voltammetric behavior of dopamine does not change significantly upon complexation, its chronoamperogram is changed due to the formed complex. The analysis was performed by titrating dopamine (guest) with variable amounts of β-cyclodextrin (host). In each mole ratio of host : guest, the solution was subjected to a potential step experiment, where in each step the current was measured as a function of time. Thus, for each potential a two-fold array of current data with molar ratio in one direction and time in another direction (data matrix) was obtained. The data matrices collected at different potentials were simultaneously analyzed by MCR-ALS to earn thermodynamic parameters (e.g., stoichiometric ratio and formation constant) of cyclodextrin–dopamine complex. This is the first report of simultaneous analysis of potential step-chronoamperometric data matrices by MCR-ALS.

Magnetic microbead-based enzyme immunoassay for ovalbumin using hydrodynamic voltammetry and fluorometric detection by Hideki Kuramitz; H. Brian Halsall; William R. Heineman (pp. 1783-1789).

A paramagnetic microbead-based enzyme immunoassay was demonstrated for detecting ovalbumin (OVA). The immunoassay sandwich was made by attaching a biotinylated antibody to the streptavidin coated beads as a mobile solid phase, capturing antigen, and then exposing the antigen to an antibody conjugated with β-galactosidase. β-Galactosidase converts p-aminophenyl galactopyranoside (PAPG) and fluorescein di-β-d-galactopyranoside (FDG) to p-aminophenol (PAP) and fluorescein, respectively. The current response of PAP generated by the enzymatic reaction of β-galactosidase was detected with hydrodynamic voltammetry in a droplet using a rotating disk electrode (RDE) system. The performance of this electrochemical assay was compared with fluorometric detection of fluorescein produced by the same assay system. The limits of detection for OVA determined by hydrodynamic amperometry and fluorometry were 2.1 nM (43 fmol) and 2.9 nM (58 fmol), respectively. Furthermore, the effects of the conditions commonly found in drinking water supply systems on the OVA assay were also evaluated.

Assessment of a COD analytical method based on the photoelectrocatalysis of a TiO2 nanotube array sensor by Hongchong Chen; Jialing Zhang; Quanpeng Chen; Jinhua Li; Di Li; Chaoping Dong; Yanbiao Liu; Baoxue Zhou; Shuchuan Shang; Weimin Cai (pp. 1790-1796).

This work mainly describes assessment of a photoelectrocatalytic method to determine chemical oxygen demand (COD) in refractory and low-concentration organics using a highly effective TiO2 nanotube array sensor in a thin-cell reactor. Twenty organic compounds, including recalcitrant organics from six categories, were used as model compounds to evaluate the accuracy of the method. The correlation between theoretical oxygen demand (ThCOD) and response COD was studied. The linear regression equation COD = α

LC-MS based assay method for DPP-IV inhibitor screening and substrate discovery by Jingjing Liu; Xueheng Cheng; Lei Fu (pp. 1797-1805).

The traditional methods for dipeptidyl peptidase IV (DPP-IV) inhibitor screening using fluorescence and chromogenic detections have a number of limitations. Interference with assay readout may occur if compounds have strong absorption at the wavelength region used for the detection. Some commonly used fluorescent and chromogenic DPP-IV substrates have poor aqueous solubility and require organic solvents such as DMSO for solubilization. The use of organic co-solvents in enzymatic assays for DPP-IV may lead to unreliable results. Furthermore, some fluorescent and chromogenic DPP-IV substrates are unstable in aqueous solution and undergo hydrolysis in the absence of DPP-IV. We have developed an LC-MS based method for DPP-IV activity assay which allowed the use of wider selections of substrates than the fluorescent and chromogenic methods. The LC-MS method was validated with several known DPP-IV inhibitors in comparison with a chromogenic assay method and was used to test library compounds to discover new inhibitors of DPP-IV. In addition, being a more universal detection technology, LC-MS method facilitates the discovery of new substrates. A mini-library of tripeptides was synthesized and screened for the discovery of new substrates with improved properties for DPP-IV assay.

Identification of new dianthrone glycosides from Polygonum multiflorum Thunb. using high-performance liquid chromatography coupled with LTQ-Orbitrap mass spectrometry detection: a strategy for the rapid detection of new low abundant metabolites from traditional Chinese medicines by Wen Xu; Jing Zhang; Zhihai Huang; Xiaohui Qiu (pp. 1806-1812).

A simple, rapid method was developed using on-line high-performance liquid chromatography (HPLC)/electrospray ionization (ESI)-linear ion trap-Orbitrap hybrid mass spectrometry (LTQ-Orbitrap) to analyze bioactive constituents in the extract of Polygonum multiflorum Thunb., a traditional Chinese medicine (TCM). The qualitative analysis was carried out both in the negative and positive mode to acquire accurate mass data in full scan and data dependent MSⁿ scan. Twenty eight new dianthrone glycosides were characterized and their fragmentation behaviors were first exploited by our LC-HR-MSⁿ method. The identification and structure analysis of these chemicals provided essential data for further phytochemical studies of P. multiflorum. Moreover, the results of the present investigations indicated the relevance and usefulness of the combination of HPLC and LTQ-Orbitrap MS to detect and identify new natural products.

On-line analysis of sulfonamides in pharmaceutical wastewater based on magnetic molecularly imprinted polymer extraction and near infrared spectroscopy by Qingbo Fu; Xinping He; Guannan Lin; Hui Suo; Chun Zhao (pp. 1813-1819).

Pharmaceutical wastewater with a high content of antibiotic residuals can greatly inhibit the activity of microorganics. Dynamic monitoring of the dissolved antibiotic content is desirable to maintain optimal biodegradation conditions. For this purpose, an on-line analysis method for sulfonamides in pharmaceutical wastewater was developed by using magnetic molecularly imprinted polymers (MMIPs) extraction and near infrared spectroscopy. An auto-sampling device was designed to perform the extraction procedure. The MMIPs synthesized had high affinity and selectivity to target sulfonamides. The automatic extraction process, including condition, extraction, washing and elution, could be carried out in several minutes, resulting in the analyte transferring from complex matrix (wastewater) to acetonitrile and thus being concentrated. The recoveries achieved ranged from 76.3% to 86.8% with relative standard deviations of 3.6% to 7.9%. Partial least squares regression was used for modeling and the proposed model predicted R² = 0.977 for the HPLC method and the absolute residuals of prediction were less than 5 mg L⁻¹. This method can rapidly provide a reference for the drug content and is promising for on-line analysis of sulfonamides in pharmaceutical wastewater. By changing the template molecule of MMIPs, it can also be used for the determination of other antibiotics.

Comparison of matrix solid-phase dispersion and modified QuEChERS methods for extraction of pesticide residues from onion by Sherol Acosta Rodrigues; Sergiane Souza Caldas; Márcia Helena Scherer Kurz; Liziara da Costa Cabrera; Fábio Andrei Duarte; Renato Zanella; Ednei Gilberto Primel (pp. 1820-1824).

Fabrication of a fast, simple and sensitive voltammetric sensor for the simultaneous determination of 4-aminohippuric acid and uric acid using a functionalized multi-walled carbon nanotube modified glassy carbon electrode by Jahan Bakhsh Raoof; Reza Ojani; Mehdi Baghayeri; Farzaneh Ahmadi (pp. 1825-1832).

In the present study, for the first time, a simple and rapid electrochemical sensor was developed for the simultaneous determination of 4-aminohippuric acid (PAH) and uric acid (UA), based on the excellent properties of functionalized multi-walled carbon nanotubes (fMWCNTs). A glassy carbon electrode was modified via drop casting the suspension of fMWCNT onto its surface and then the modified electrode was used for the measurement. The electrochemical response characteristics of the modified electrode toward PAH and UA were investigated using cyclic voltammetry, differential pulse voltammetry and chronoamperometry. The values of transfer coefficient for oxidation of PAH and UA were calculated using electrochemical approaches and were found to be 0.43 and 0.46, respectively. PAH and UA in a mixture can be measured individually and simultaneously by differential pulse voltammetry. At the optimum pH of 6.0 in 0.1 M phosphate buffer solution, differential pulse voltammograms increase linearly with PAH and UA concentrations over the concentration ranges of 10–1000 μM of PAH and 1–100 μM of UA. The detection limits for PAH and UA were found to be 7.82 μM and 0.96 μM, respectively. The proposed method was also successfully applied to the determination of PAH and UA in real samples.

High performance liquid chromatography (HPLC) method coupled with resonance Rayleigh scattering detection for the determination of isepamicin by Jinxia Tang; Jingdong Peng; Lei Zhang; Ying Xiao (pp. 1833-1837).

A simple and novel reversed-phase high performance liquid chromatography (HPLC) method coupled with resonance Rayleigh scattering (RRS) was developed to analyze isepamicin (ISP) in rat plasma. The chromatographic separation was achieved at 30 °C on a reverse phase column of Synergi Hydro-RP (150 mm × 4.6 mm; 4 μm), with (15 : 85, v/v) methanol–water (containing 0.15% TFA (v/v)) as the mobile phase. The flow rate was 0.4 mL min⁻¹. The RRS signal was detected at λex = λem = 372 nm. Netilmicin (NTL) was used as the internal standard. The new analytical technique was validated for the intra- and inter-day variation, the linear range of the standard curve of ISP and the limit of detection (S/N = 3). The results indicated that HPLC coupled with RRS was a reliable and valuable technique for quantitative analysis of ISP in rat plasma. The method shows a better linear relationship and lower analysis time.

Potentiometric glucose biosensing using camphor sulfonic acid doped polyaniline by T. Sowmiya; A. Ananthi; S. Anandhakumar; J. Mathiyarasu (pp. 1838-1842).

In this work, we explored the possibility of developing an enzymatic potentiometric glucose biosensor using polyaniline–camphorsulfonic acid–glucose oxidase (PANI–CSA–GOD) composite films. Glucose oxidase was covalently immobilized on the polyaniline–camphorsulfonic acid modified electrode that was used as a potentiometric sensor matrix. The PANI–CSA–GOD transducer responded linearly to glucose molecules in the concentration range of 1 to 50 mM and the sensitivity was found to be 0.09 mV mM⁻¹. The modified electrode showed excellent sensitivity and better selectivity in the presence of some common interfering molecules.

Back cover (pp. 1843-1844).

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Analytical Methods (v.4, #6)

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Analytical Methods (v.4, #6)