Analytical Methods (v.4, #4)

Front cover (875-875).

Inside front cover (876-876).

Contents list (877-892).

Robust regression: An introduction by Analytical Methods Committee, AMCTB No 50 (893-894).
Analytical scientists use regression methods in two main areas. Calibration graphs are used with the results of instrumental analyses to obtain concentrations from test samples. Graphical methods are used to evaluate the results obtained when two methods, often a novel one and a reference one, are compared by applying them to the same set of test materials. In either case outliers or suspect results may occur, and exert big effects on the plotted regression line and the results derived from it. Robust methods are well suited to tackling such situations. Here some of the underlying ideas are summarised: later briefs will describe some more of the many approaches available.

G-quadruplex fluorescence quenching ability: a simple and efficient strategy to design a single-labeled DNA probe by Xiaohai Yang; Ying Zhu; Pei Liu; Leiliang He; Qingzhao Li; Qing Wang; Kemin Wang; Jin Huang; Jianbo Liu (895-897).
A single labeled fluorescence probe was designed based on the efficient quenching ability of a G-quadruplex instead of traditional quenchers. Using this probe, we have investigated the quenching ability of a G-quadruplex and developed a new approach to analyze mercury ions and cysteine with high specificity and sensitivity. Owing to the high quenching ability of the G-quadruplex and the fidelity of the “thymine–Hg2+–thymine” binding motif, this approach can detect 4.0 nM mercury ions, which is lower than the 10 nM US EPA limit in drinking water.

A water-soluble bidentate pyridine–acid ligand (1) was synthesized. The ligand 1 had a stronger interaction with Mo6+ than with other metal ions. Subsequently, we found that a linear increase in the fluorescence intensity of complex (1-Mo) was observed upon addition of bovine serum albumin (BSA).

Quality assurance in immunoassay performance-temperature effects by Julia Grandke; Ute Resch-Genger; Wolfram Bremser; Leif-Alexander Garbe; Rudolf J. Schneider (901-905).
Temperature is one factor that influences the performance of immunoassays. Most commonly all incubation steps of the immunoassay are performed at ambient temperature. To systematically study the influence of temperature deviations on assay parameters like signal variation coefficients, limit of detection and measurement range, direct competitive enzyme immunoassays for the determination of two anthropogenic markers, caffeine and carbamazepine, were run at different temperatures above and below room temperature during individual assay steps. The temperature during the competition step had the greatest impact on the assay sensitivity yielding changes in test midpoints by a factor of 10 to 15. For carbamazepine, the test midpoints varied from 36 ng L−1, 108 ng L−1 to 378 ng L−1 employing 4 °C, 21 °C, or 37 °C as incubation temperature, respectively. The edge effect can be minimized resulting in a good plate homogeneity with small variation coefficients, when the assay is performed at ambient temperature.

Separation of enantiomers, dansyl-dl-amino acids, was carried out by open-tubular capillary chromatography based on the tube radial distribution of the carrier solvents. An untreated poly(tetrafluoroethylene) capillary tube (100 μm inner diameter and 90 cm effective length) as a separation column and a water–acetonitrile–ethyl acetate mixture containing cyclodextrin as a carrier solution were used in the chromatography. An analyte solution of dansyl-dl-amino acids, such as dansyl-dl-methionine, was injected into the capillary tube by a gravity method. The analyte solution was subsequently delivered through the capillary tube with the carrier solution by a microsyringe pump. The ternary mixed carrier solution (water-rich carrier solution) was radially distributed in the capillary tube based on the tube radial distribution phenomenon, causing the formation of inner (water-rich) and outer (organic solvent-rich) phases. The outer or capillary wall phase acted as a pseudo-stationary phase in the chromatography. The analytes were separated through the capillary tube with on-capillary detection by an absorption or a fluorescence detector. The d-enantiomer and the l-enantiomer were eluted in this order with a baseline separation. The separation mechanism of the enantiomers in the open-tubular capillary using cyclodextrin was discussed.

Fluorescent boronic acid-modified polymer nanoparticles for enantioselective monosaccharide detection by Wan-Kyu Oh; Yoon Seon Jeong; Kyung Jin Lee; Jyongsik Jang (913-918).
We report the fabrication of new fluorescent boronic acid-modified polyacrylonitrile (B-PAN) nanoparticles for an enantioselective monosaccharide sensor. Polyacrylonitrile (PAN) nanoparticles of 50 nm diameter were synthesized by radical polymerization. After boronic acid modification, the B-PAN nanoparticles showed enhanced fluorescence due to a photo-induced electron transfer mechanism. The B-PAN nanoparticles enabled molecular recognition in aqueous solution owing to covalent bonds with diol-containing compounds, resulting in application for enantiomer detection of monosaccharides. The fluorescence intensity changed when B-PAN nanoparticles interacted with the enantiomers of monosaccharides, including d-/l-glucose, d-/l-galactose, and d-/l-fructose. The B-PAN nanoparticles provide a new direction for the development of enantioselective monosaccharide sensors and could be subsequently expanded to a platform of versatile fluorescence sensors.

Copper-promoted probe for nitric oxide based on o-phenylenediamine: Large blue-shift in absorption and fluorescence enhancement by Xiaolong Sun; Yufang Xu; Weiping Zhu; Chunsheng He; Lin Xu; Youjun Yang; Xuhong Qian (919-922).
We developed a new o-phenylenediamine-based compound as a turn-on fluorescent probe for nitric oxide (NO) with a large blue shift in absorption. It showed high selectivity and sensitivity toward nitric oxide over other ROS and RNS species in neutral buffer solution. The analytical detection limit (ADL) was 30 nM. As far as we are aware this is the first o-phenylenediamine-based NO probe using Cu2+ as a promoter.

A new methodology is developed for the accurate detection of precipitation titration end points without using any indicator. The validity of this new scanometry method was studied by detection of end points of the titration of halides in single, binary and ternary solutions. The cell containing precipitate was scanned with a flatbed scanner, and the image was analyzed in the CMYK color model by a written color analysis program. The results revealed that among the color values, the black value was suitable for the accurate detection of end points. To improve the accuracy of the end point detection, second derivative titration curves were obtained. The titration errors were acceptable, and were always less than 10%. The possibility of qualitative detection of end points, without using any image analysis, was also studied. The results indicated that in some cases, the end points could be visually detected from the cell image without applying any color analysis.

A thin layer chromatographic method combined with an image processing system (MIA-TLC) was developed for quantitative monitoring of the progress of organic reactions. Two simple reactions including alkaline hydrolysis of phenyl benzoate and reduction of benzaldehyde derivatives were chosen as model reactions. Small aliquots of starting material as well as successive aliquots of the reaction mixture in the course of the reaction were spotted on the TLC plate by micropipette. Hexane–ethyl acetate (with volume ratios of 15 : 1 and 3 : 1 for alkaline hydrolysis and reduction reactions, respectively) was chosen as the mobile phase. After TLC development, images of the plates were recorded using a home-made imaging cabinet. A home-made program was able to covert image data into three and two dimensional chromatograms, from which changes in the amount of reactants as a function of reaction time was easily monitored. By analysis of obtained chromatograms as a function of reaction time we calculated the conversion percentage of the reactions. The conversion percentage for both reactions was almost 99.9%. Also, the data enabled us to monitor the kinetics of the reactions and estimate the rate constants. Conversion of visually interpretable information to color values for quantitative analysis by MIA-TLC is a rapid and inexpensive method recommended as a powerful tool for quantitative reaction monitoring as well as a good alternative for GC in organic laboratories.

This paper attempted to show the feasibility of measuring the antioxidant activity in dark soy sauce by NIR spectroscopy technique. Chemometrics on spectral intervals selection and nonlinear regression tools were systematically studied in the calibrating model. First, the optimal spectral intervals were selected by synergy interval-partial least square (Si-PLS). Then, kernel PLS (KPLS) and back propagation artificial neural network (BPANN), as two nonlinear regression tools, were performed comparatively to calibrate models based on optimal spectral intervals, called Si-KPLS and Si-BPANN models, respectively. These models were optimized by cross-validation, and the performance of the final model was evaluated according to correlation coefficient (Rp2) and root mean square error of prediction (RMSEP) in the prediction set. The results showed that the Si-BPANN model was superior to other models, and the optimal result was achieved with Rp2 = 0.9769 and RMSEP = 0.0221 in the prediction set. This work demonstrated that total antioxidant capacity in dark soy sauce could be measured by NIR spectroscopy technique, and Si-BPANN showed its superiority in model calibration.

Determination of magnesium ion in serum samples by a DNAzyme-based electrochemical biosensor by Xiaoyao Gao; Huiming Huang; Shuiying Niu; Huazhen Ye; Zhenyu Lin; Bin Qiu; Guonan Chen (947-952).
A DNAzyme-based electrochemical biosensor for magnesium ion (Mg2+) has been developed. Firstly, the Mg2+-dependent DNAzyme with thiol at the 5′-terminus is immobilized on a gold electrode through thiol–Au interaction. Then the substrate strand with ferrocene (Fc) at the 5′-terminus is attached to the gold electrode via hybridization with DNAzyme. In the absence of Mg2+, this sensor shows an obvious faradaic current from Fc. In the presence of Mg2+, the Fc-labeled substrate strand is cleaved into two pieces by catalytic action of DNAzyme and moves away from the electrode surface, which reduces the faradaic current. The current change provides quantitative measurement of Mg2+. The linear response range for the proposed biosensor is 0.2–5.0 mmol L−1, and the detection limit is 0.05 mmol L−1. Other divalent metals present do not interfere with Mg2+ detection. The sensor was applied to detect Mg2+ in serum with satisfactory results.

Electrochemical behavior of para-nitroaniline at a new synthetic crown ether-silver nanoparticle modified carbon paste electrode by Mohammad Hossein Arbab Zavar; Somayeh Heydari; Gholam Hossein Rounaghi; Hossein Eshghi; Hossein Azizi-Toupkanloo (953-958).
A new synthetic ligand (6,7,9,10,17,18,19,20,21,22-decahydrodibenzo[h,r][1,4,7,11,15] trioxadiazacyclonanodecine-16,23-dione, DTD)–Ag nanoparticles (AgNP) modified carbon paste electrode (DTD/Ag–CPE) is fabricated. Voltammetric behavior and determination of para-nitroaniline (PNA) is explored. The influence of various experimental parameters on the electrode response is investigated. Under optimized conditions the peak current showed a good linear relationship with p-nitroaniline concentration in the range 1 × 10−6 mol L−1 to 1 × 10−4 mol L−1. The proposed electrode was applied to p-nitroaniline determination in water samples.

In this paper, new modified magnetite nanoparticles functionalized with triazene groups were designed and synthesized for extraction/preconcentration of sub-ppb level of mercury ions in water and fish samples prior to its determination with inductively coupled plasma optical emission spectrometry (ICP-OES). In the separation process, aqueous solution of Hg2+ ions was mixed with 150 mg of Fe3O4 magnetite nanoparticles modified with 1-(p-acetyl phenyl)-3-(o-ethoxy phenyl) triazene (AET) and then external magnetic field was applied for isolation of magnetite nanoparticles containing mercury ions. Experimental conditions for effective adsorption including pH, sample volume, eluent concentration and existing co-existing ions have been studied and established. Under the optimal extraction and preconcentration conditions, the limit of detection (LOD) of 0.04 ng mL−1 and the relative standard deviation (R.S.D) of 2.09% for five replicate extractions and measurements of 10 μg of Hg2+ ion in 1000 mL water solution were achieved by ICP-OES. The sorption capacity of functionalized Fe3O4 magnetite nanoparticles under optimum conditions has been found to be 10.26 mg of mercury ion per gram at pH 7 with the preconcentration factor of 500 (2 mL of elution for a 1000 mL sample volume). Standard solutions containing Hg2+ in the concentration range of 0.2–200 ng mL−1 were examined by the proposed procedure and it was observed that calibration curve was linear in this range (R2 = 0.999). The special advantages of the proposed method are high enrichment factor, fast separation and low detection limits compared with other methods.

Development of an electrochemical DNA biosensor for direct detection and discrimination of HCV core/E1 region corresponding double-stranded DNA (ds-DNA) using a peptide nucleic acid (PNA) oligomer as the probe is described. The PNA probe is a cysteine conjugated 20-mer PNA oligomer, complementary to HCV core/E1 universal region, which is a consensus sequence in almost all HCV genotypes and is not present in other organisms. The significant variation in differential pulse voltammetric response of methylene blue (MB) on the probe modified gold electrode (AuE) upon hybridization with complementary double-stranded oligonucleotide (ds-oligonucleotide) following PNA/ds-DNA hybrid formation is the principle of target ds-DNA detection. No significant variations in MB signal following interaction of the probe with non-complementary and single-base mismatch (SBM) ds-DNAs was observed. This is due to the lack of hybridization between the probe and the non-complementary and SBM ds-DNA samples. Diagnostic performance of the biosensor is described and the detection limit of fully match target ds-DNA was found to be 9.63 × 10−12 M and 4.97 × 10−12 M for 2 h and 20 h hybridization times, respectively. The relative standard deviation over three independently probe modified electrodes measured at 100 pM of target ds-DNA was 2.9% and 2.4% for 2 h and 20 h hybridization times indicating a remarkable reproducibility of the detection method.

Simultaneous determination of acetaminophen, propyphenazone and caffeine in cefalgin preparation by pressurized planar electrochromatography and high-performance thin-layer chromatography by Aneta Hałka-Grysińska; Piotr Ślązak; Grzegorz Zaręba; Wojciech Markowski; Anna Klimek-Turek; Tadeusz H. Dzido (973-982).
Pressurized planar electrochromatography (PPEC) and high-performance thin-layer chromatography (HPTLC) have been involved in separation and analysis of active components of cefalgin tablets: acetaminophen, propyphenazone and caffeine. A separation system comprised acetonitrile-buffer mobile phase and chromatographic plates with an adsorbent layer of the C18 type. The PPEC separation process, applying polarization voltage 1.8 kV, was 2.7 times shorter than that of HPTLC. Total time of the separation procedure by PPEC mode (6 min) is considerably shorter than that of HPTLC (20 min). Resolution of sample bands and performance of the PPEC separating system are more favorable for quantitative analysis than those of HPTLC under the same chromatographic conditions (qualitative and quantitative composition of the mobile and stationary phases). Under established experimental conditions, satisfactory sensitivity of the method was achieved. The LODs ranged from 0.06 to 0.16 μg of component per spot depending on the method and substance. Procedures were validated for selectivity, precision, linearity and accuracy. Investigations show that both PPEC and HPTLC modes were accurate, precise, sensitive, linear and specific. Furthermore, PPEC is promising mode, which, in our opinion, can be applied in routine pharmaceutical analysis in the near future.

The analysis of triazine and phenylurea herbicides in soil by ionic liquid based microwave-assisted extraction (IL-MAE) coupled with high-performance liquid chromatography was studied. The operational IL-MAE parameters were optimized, including types and concentration of ionic liquid, microwave power, irradiation temperature, extraction time and extractant volume. The results obtained under the optimized conditions were compared with methanol-MAE and IL-heating extraction, and it was determined that the IL-MAE method was rapid (4 min compared with 2 h for the heating extraction), efficient (recoveries were 1.6–12.2% higher than the other methods) and environmentally friendly (no volatile organic solvents were used). The proposed analytical method was evaluated by considering the average spike recoveries (84.0–101%) and relative standard deviations (2.8–6.0%), all of which indicate that the IL-MAE method is accurate, reproducible, and could be applied to real sample analyses.

Determination of lead in water by combining precolumn adsorption and fluorimetric detection in a microfluidic device by Ting Wu; Liyun Zhao; Djibril Faye; Jean-Pierre Lefevre; Jacques Delaire; Isabelle Leray (989-994).
A microfluidic device equipped for in situ fluorescence detection and combined with an off-line microcolumn adsorption is proposed for the determination of lead in natural water. The precolumn adsorption is based on a microcapillary filled with aminopropyl silica. An efficient Pb2+ adsorption on aminopropyl silica was proved by Langmuir isotherm adsorption experiments. The maximum adsorption capacity was found to be 5.7 × 10−4 mol g−1. By using this solid phase extraction procedure (SPE), a 100-fold specific lead concentration was obtained. The concentrated solution was then run into a microfluidic device equipped for fluorescence detection and the complete procedure allows lead determination with a detection limit of 2 μg L−1. Furthermore, it was found that these measurements are not disrupted by the addition of a large excess of other interfering cations.

A new ultrasound-assisted extraction (UAE) followed by dispersive liquid–liquid microextraction (DLLME) method has been developed for the simultaneous determination of flavoring compounds, including safrole, coumarin, 6-methylcoumarin, 7-metheoxycoumarin, estragole, methyleugenol, pulegone and thujone, in tobacco additives. In this method, the targets were extracted from tobacco additive sample using ethanol by UAE. Then, 0.5 mL of the extract were used for the DLLME. In the DLLME, the target analytes in the extraction solvent were rapidly extracted into a small volume of chloroform and analyzed by chromatography-mass spectrometry (GC-MS). The parameters affecting the DLLME, including type and volume of extraction and disperser solvents, salt effect and extraction time, were investigated and optimized. Under the optimum conditions, the enrichment factors (EFs) ranged from 140 to 208. The linear relationship was obtained in the range of 0.4–928 ng mL−1, which showed satisfactory linearity with correlation coefficients (r2) over 0.9989 for all the analytes. The limits of detection (LOD) were between 0.04 and 0.24 ng mL−1 and the average recoveries were between 89.9 and 99.7% with relative standard deviations (RSDs) ranging from 2.5 to 6.7% in all cases. The method was successfully applied to the preconcentration and determination of these target compounds in tobacco additives.

Preparation of novel core-shell silica particles for pH sensing using ratiometric fluorescence approach by Ting-xiu Ye; Yan-yan Du; Chun-yan He; Bin Qiu; Yi-ru Wang; Xi Chen (1001-1004).
Core-shell silica particles encapsulating two luminescent dyes with different emission wavelengths were prepared for ratiometric fluorescence sensing of pH. The composite core-shell structure particles contained a polyacrylonitrile (PAN) core surrounding a silica shell. Fluorescein isothiocyanate (FITC) encapsulated in the silica shell was taken as an indicator, Fluorescence emission from Ru(dpp)3(ClO4)2 immobilized in PAN core was used as a reference light. The ratiometric fluorescence signal from the core-shell silica particles were found with a linear range from pH 5.80 to 7.50 (R2 = 0.997). In addition, the morphology of the core-shell silica particles, the effects of temperature and co-existing substances were investigated. The core-shell silica particles were successfully applied to determination of pH value in water and urine samples.

Novel magnetic molecularly imprinted polymers were prepared using bilayer modified Fe3O4 magnetite as the magnetically susceptible component, oxytetracycline and chlortetracycline as the mixed-template molecules, and methacrylic acid as the functional monomer. The magnetic polymers were applied to the separation of tetracycline antibiotics from egg and honey samples. The sample, polymers and extraction solvent were blended and stirred together. The extraction and clean-up procedures were carried out in a single step. The analytes could be extracted from the sample matrix to the polymers directly or through the extraction solvent as the medium. When the extraction was complete, the polymers which had adsorbed the analytes were easily separated from the sample matrix by an adscititious magnet. The analytes eluted from the polymers were determined by high liquid chromatography spectrometry. The recoveries obtained to be 76.2% to 95.8% with relative standard deviations in the range of 3.1–13.2%. The limit of detection was less than 0.2 ng g−1. The feasibility of this method was proven by analysis of incurred egg and honey samples. Compared with the classical method which uses more steps including solvent extraction, centrifugation, clean-up and concentration by solid-phase extraction, the proposed method decreased the complexity and improved the reliability.

Application of ionic liquids in the microwave-assisted extraction of quercetin from Chinese herbal medicine by Xiaojie Liu; Yuzhi Wang; Jinhuan Kong; Chan Nie; Xiao Lin (1012-1018).
Eight types of ionic liquid (IL) were synthesized in this paper, and ionic liquids-based microwave-assisted extraction (ILs-MAE) technique was used to extract quercetin from the Chinese herbal medicine Toona sinensis. Several significant operating parameters were optimized by a series of single factor experiments and orthogonal design L9 (34) experiments in this work. Compared with liquid-based maceration extraction (ILs-ME), liquid-based heat extraction (ILs-HE) and liquids-based ultrasound-assisted extraction (ILs-UAE), the optimized approach of ILs-MAE technique cost the shortest time but afforded the highest extraction amount. Moreover, reversed phase high performance liquid chromatography (RP-HPLC) with ultraviolet detection (UV-DAD) was employed for the analysis. The results show that the amount of quercetin extracted by [Bmim]Br was 182.78 mg g−1 with RSD 1.02%. The developed approach is linear between concentrations from 15.20 mg L−1 to 213.2 mg L−1 of quercetin and the regression coefficient (R) was 0.9992. The recoveries of quercetin extracted by these four methods were 95.07%–105.8% with RSDs lower than 3%. Furthermore, the change of the microstructure and chemical structure of the sample before and after different extraction procedures were researched, respectively. Because of the remarkable dissolving capacity of ILs aqueous solution and the obvious destruction on sample surface microstructures during ILs-MAE process, ILs-MAE technique got the highest extracted amount. With the help of LC-MS, we can further confirm that Toona sinensis do contain the active ingredient of quercetin and ILs had no interference of quercetin.

DRDE-07 is a newly synthesized amifostine analog found to be very effective as a prophylactic agent against sulphur mustard toxicity. It has been proven that orally administered DRDE-07 is more efficacious than amifostine, against percutaneously administered SM. We validated an analytical protocol for DRDE-07 in mice urine using high performance liquid chromatographic method. Heptane sulphonic acid and tetramethyl ammonium chloride were used in the mobile phase as an ion pairing agent to perform the chromatographic separation. UV detection was carried out at 249 nm, a wavelength at which an absorption peak was detected. The calibration curve for DRDE-07 was linear in the range from 1 to 100 μg ml−1. The lower limit of quantification (LLOQ) was 1 μg ml−1. The results demonstrate that this method has high linearity (R2 = 0.9986), compound specificity, and acceptable precision/accuracy. The protocol is suitable for in vivo determination of DRDE-07 in mice urine.

Direct electron transfer of ferritin on electrodeposited nickel oxide cubic nanoparticles by Ali Mohammadi; Abdolmajid Bayandori Moghaddam; Jalil Badraghi (1024-1028).
Immobilization of protein molecules on the surface of nanoparticles has many applications in sensing and biosensing. A variety of covalent and non-covalent methods have been reported for protein immobilization on nanoparticles. In this work, nickel oxide cubic nanoparticles (NiOx NPs) were synthesized by electrodeposition method. Iron storage protein molecules (ferritin) were directly adsorbed on the surface of the nanoparticles by cyclic voltammetry (CV). Electrochemical parameters of immobilized ferritin including formal potential (E0′) and charge transfer coefficient (α) were estimated. The results show that the electrodeposited NPs provide an efficient substrate for the immobilization of ferritin. The prepared biosensor exhibits an electrocatalytic activity for the reduction of hydrogen peroxide.

Application of a modified carbon nanotube paste electrode for simultaneous determination of epinephrine, uric acid and folic acid by Hadi Beitollahi; Alireza Mohadesi; Saeedeh Khalilizadeh Mahani; Ali Akbari (1029-1035).
A novel 5-amino-3′,4′- dimethoxy-biphenyl-2-ol (5ADMB) modified carbon nanotube paste electrode (5ADMBCNPE) was first prepared for the determination of epinephrine (EP). Cyclic voltammetry (CV) was used to investigate the electrochemical properties of the proposed electrode towards the oxidation of EP. It has been found that under an optimum condition (pH 7.0), the oxidation of EP at the surface of the modified electrode occurs at a potential of about 160 mV less positive than that of an unmodified carbon paste electrode. The diffusion coefficient (D = 1.18 × 10−5 cm2 s−1), and electron transfer coefficient (α = 0.53) of EP at the surface of 5ADMBCNPE were determined using electrochemical approaches. Using square wave voltammetry (SWV) a linearity between the oxidation peak current and the EP concentration was obtained in the range of 0.4 to 900.0 μM with a detection limit (3σ) of 88.0 nM. Also, SWV was used for simultaneous determination of EP, uric acid (UA) and folic acid (FA) at the modified electrode.

Impedimetric DNA sensor for detection of Hg2+ and Pb2+ by Liu Shi; Gang Liang; Xiaohong Li; Xinhui Liu (1036-1040).
An electrochemical DNA sensor based on DNA conformational changes for simultaneous detection of Hg2+ and Pb2+ was reported. The sensor was consisted of a probe strand (DNA), a Pb2+-specific DNAzyme, and a substrate strand contains mercury-specific oligonucleotide (MSO). When Hg2+ and Pb2+ interacted with DNA, the induced conformational changes were tracked by electrochemical impedance spectroscopy (EIS), which led to a decreased RCT. The RCT difference (ΔRCT) was applied to selectively detect Hg2+ and Pb2+ with detection limit of 1 pM and 0.1 pM, respectively. Through using masking agents, such as cysteine (masking Hg2+) and G-DNA (CTG-GGA-GGG-AGG-GAG-GGA) (masking Pb2+), Hg2+ and Pb2+ were simultaneously detected in buffer solution, human serum and river water, respectively.

The identification of the 6-benzylaminopurine (6-BAP) plant growth regulator was performed using a label-free opto-fluidic ring resonator (OFRR) biosensor. In this study, the OFRR biosensor with a BRIS sensitivity of 47.6 nm/RI was fabricated through the optimization of the manufacturing process of the OFRR capillary and a tapered fiber optic cable, which are the key components of the OFRR biosensor. The minimum detection concentration of 6-BAP standard solution and 6-BAP extracted from bean sprouts using QuEChERS method were 0.05 ppm in the fabricated OFRR biosensor system. In addition, the response signal was amplified up to sixfold through the application of a one-step sandwich assay that uses gold nanoparticles (AuNP) conjugated with anti-6-BAP to improve the measuring sensitivity of 6-BAP. As a result, the minimum detection concentration of 6-BAP was lowered to 0.01 ppm and it was possible to detect more segmented WGM spectral changes at lower concentrations.

Near-infrared (NIR) spectroscopy combined with the moving window partial least-squares (MWPLS) method and Savitzky–Golay (SG) smoothing was successfully applied for the rapid no-reagent determination of chemical oxygen demand (COD) in sugar refinery wastewater. An appropriate waveband with stability was selected through a rigorous modeling process. Among 141 samples, 60 were randomly selected as the validation set. The remaining 81 samples were divided into the calibration set (50 samples) and the prediction set (31 samples) for a total of 20 times with certain similarities. The results showed that the short-wave NIR region (780 nm to 1100 nm) can be used as the information waveband of COD in sugar refinery wastewater, and the optimal SG smoothing mode was 5th order derivative, 5th degree polynomial, and 7 smoothing points. The waveband selection was performed in the SG smoothing spectra of the short-wave NIR region by the MWPLS method. The optimized waveband was 808 nm to 964 nm, the validation root mean square error of prediction (V-SEP) and validation correlation coefficients of prediction (V-RP) were 25.0 mg L−1 and 0.966, respectively, which had good prediction precision and stability. The results provide a reliable NIR analysis model and valuable references for designing small, specialized NIR instruments.

CdS nanotubes thin film for electrochemiluminescence analysis of phenolic compounds by Yaqiong Wen; Fulian Luo; Yinling Yang; Lin Lin; Juan Du; Yong Guo; Dan Xiao; Martin M. F. Choi (1053-1059).
A CdS nanotubes (NT) thin film has been fabricated to determine phenolic compounds based on the electrochemiluminescence (ECL) quenching of the CdS NT. The CdS NT thin film coated on an indium tin oxide substrate exhibits strong ECL emission with potassium persulfate (K2S2O8) as a coreactant. The effects of pH, electrolyte concentration and cyclic voltammetric scan rate on the ECL were studied. The quenching efficiency is governed by the position of the hydroxyl moiety in the parent benzene ring which follows the trend: resorcinol (1,3-benzenediol) > catechol (1,2-benzenediol) > hydroquinone (1,4-benzenediol) ≈ phenol. Under the optimal experimental conditions of 0.010 M K2S2O8, pH 11.60 and 0.10 V s−1, the quenching of ECL by catechol (2.0–10.0 μM), hydroquinone (40.0–140 μM), phenol (10–60 μM) and resorcinol (2.0–12.0 μM) follows Stern–Volmer behaviour. The limits of detection are calculated as 0.058, 0.64, 1.7, and 0.069 μM (S/N = 3) for catechol, hydroquinone, phenol, and resorcinol respectively. The fabrication of the CdS NT film is reproducible and its shelf-life is longer than three months. The CdS NT film has been successfully applied to determine the phenol contents in various environmental water samples with satisfactory results and good recoveries. Our proposed ECL CdS NT film provides a simple and convenient method for the detection of phenolic compounds and shows potential use in the pharmaceutical industry and environmental monitoring.

Today, (bio-)analytical researchers use various software tools for improving data analysis and the evaluation of their experimental results. Bioinformaticians typically program these software tools; however, analysts and bioinformaticians have distinct views on these data. Achroma is a software tool for the analysis of spectrometric data; it is our hope that explaining the software strategy and the working modules behind Achroma may give analysts a better understanding of how (bio-)informaticians work out software solutions, thus facilitating the interaction between these two expert groups. Achroma is based on a model-view-controller (MVC) software architecture. Furthermore, Achroma has a modular structure and each module has its own MVC architecture. Typically, each module delivers just one specific function to the user. Finally, every module is embedded within Achroma as a small “stand alone” software application. Analytically, Achroma software is a tool to handle typical and untypical mass spectrometric data. Achroma was originally programmed to circumvent problems with mass spectrometric vendor software in the analysis of data from new experimental strategies. Specifically, existing software enables the data analysis from continuous-flow mixing systems monitoring enzyme–inhibitor reactions only manually and indirectly. This data-handling bottleneck is resolved in Achroma. The principles behind this implementation are described in detail for two modules—the ‘chromatogram comparison’ and ‘signal recognition’ modules. Further specific examples of data analysis are presented, such as signal recognition, chromatogram smoothing and signal area calculation. We regard such understanding between the analysts' and programmers' worlds as essential for future improvements in analytical software.

A new methodology ultrasound assisted cold-induced aggregation microextraction (USA-CIAME) was developed for the determination and extraction of ultra trace levels of Au3+ from aqueous hydrochloric acid solutions by pure ionic liquid (IL), in the absence of chelating agents. The extraction behaviour of metal ions by [Hmim]PF6 was particularly studied. Moreover, the stripping of metal ions from the IL phase was investigated. Au3+ in IL phase can be quantitatively recovered by NaPF6. Recycle testing indicated that IL loss can be avoided by the additon of [Hmim]+ to the initial AuCl4 solutions before extraction and/or using PF6 solutions for stripping. The main factors affecting Au3+ extraction, the amount of IL, the pH of the sample solutions, and the chloride ion concentration on extraction efficiencies were carefully studied. A linear calibration was obtained for 1.5 to 200 μg L−1 of Au3+ and the limit of detection was 0.59 μg L−1. The validation of this method was done by recovery and determination of trace amount of gold in river, sea water and geological samples with satisfactory results.

Screening target components from Radix salviae miltiorrhiae using an EGFR/CMC-online-HPLC/MS method by Sheng-Li Han; Tao Zhang; Jing Huang; Zhi-Gang Hu; Si-Cen Wang (1078-1083).
Radix salviae miltiorrhizae is a widely used traditional Chinese medicine, it is the root and rhizome of Salvia miltiorrhiza Bge and belongs to the family Labiatae. Many of its pharmacological effects have been reported in a previous study. In this study, a two-dimension online analytical method of epidermal growth factor receptor cell membrane chromatography combined with liquid chromatography/mass spectrometry was built for recognition, separation and identification of active components from traditional Chinese herb Radix salviae miltiorrhizae. Gefitinib was used as positive control, while atenolol, nicotine, and nimodipine were used as negative controls to investigate the selectivity of the EGFR/CMC column and system suitability of this online analytical method. Cryptotanshinone was screened from Radix salviae miltiorrhizae by this online analytical method. Through in vitro pharmacological trials, cryptotanshinone showed obvious and dose-dependent inhibition activity in the range 0.40 to 50.0 μM. Molecular docking assay showed that the cryptotanshinone binding region was in very good agreement with gefitinib. The EGFR/CMC-online-LC/MS two-dimension analytical method was demonstrated as an effective screening method that rapidly recognizes and enriches active components acting on the epidermal growth factor receptor from Radix salviae miltiorrhizae and separates and identifies them.

A strategy for both low-concentration calibration range and large number of sample sets selection in the development of a PLS model is studied. A novel approach based on accuracy profile validated the accuracy and precision of the PLS model. The strategy was applied to the determination of chlorogenic acid content in Lonicera japonica using ethanol precipitation by near-infrared (NIR) transmission spectroscopy. The results found the determination coefficient (R2), standard errors of calibration and prediction (SEC and SEP) were 0.9648, 71.2 ppm and 74.9 ppm, respectively. The further study showed that PLS model could be used to determine chlorogenic acid content based on accuracy profile, which has a lower limit of quantification (LLOQ) ∼1700 ppm. Analytical properties such as accuracy, precision, range and linearity from validation criteria also demonstrated the feasibility of the strategy using a low-concentration calibration set in the PLS model, paving the way for analyses in Chinese Herbal Medicine (CHM) applications.

Flow injection analysis for nitric oxide quantification based on reduced fluoresceinamine by Eliana F. C. Simões; João M. M. Leitão; Rui M. Barbosa; Joaquim C. G. Esteves da Silva (1089-1097).
A fluorescence flow injection analysis (FIA) methodology for nitric oxide (NO) quantification was optimized by factorial analysis for the lowest limit of detection of nitric oxide. This methodology is based on the reaction of the NO with the non-fluorescent reduced fluoresceinamine given a high fluorescent oxidized fluoresceinamine. Box–Behnken and central composite optimization experimental design methodologies were used. The factors initially analysed by a screening experimental design methodology were the flow rate of the pump (Q), loop volume (L), reactor length (R), reduced fluoresceinamine concentration (CFl) and cobalt chloride concentration (CCoCl2). The response variables under analysis were the maximum fluorescence intensity, response repeatability and peak width. The optimum conditions were: one flow stream FIA configuration, Q = 0.60 mL min−1, L = 100 μL, R = 2 m, CFl = 1.50 mM and without CoCl2. A linear working range between 5 to 40 μM was evaluated with a limit of detection of 1.20 μM. Hydrogen peroxide, superoxide, nitrite and nitrate did not interfere with the NO detection. Good results were found in the quantification of NO liberated by a NO donor at pH 7.4 and in fortified serum samples.

A strong cation exchange monolithic column was prepared by a single step in situ radical polymerization without further chemical modification for high performance liquid chromatography (HPLC). The novel monolithic column used vinyl ester resin as the monomer, ethylene glycol dimethacrylate (EDMA) as the cross linking agent, 2,2′-azobisisobutyronitrile (AIBN) as the initiating agent, NaHSO3 both as inorganic adjunct and coadunate initiator (L. G. Bai, H. Y. Liu, Y. K. Liu, J. Chromatogr. A, 2010, 1218, 100-106, and F. A. Zhang, C. L. Yu, X. H. Zhu, C. Wei, Polym. Adv. Technol., 2006, 17, 608–611). Moreover, NaHSO3 can supply sulfonic acid groups as cation exchangers for ion-exchange monolithic column. Polymerization conditions and separation conditions were optimized, and the structure properties of the monoliths were also investigated. In addition, the chromatographic performance of the monolith was evaluated through separating human serum albumin (HSA) from human plasma in conjunction with HPLC and the expected results were acquired. The effects of buffer concentration and pH on the separation of HSA were investigated. Furthermore, the monolith was used to separate the mixture of protein (hemoglobin and HSA) with good resolution.

In this paper, a new kind of electrode for sensing hydrogen peroxide (H2O2) was reported. Silver microcrystals were initially deposited on a nichrome substrate by cyclic voltammetry and controlled potential electrolysis, respectively. Scanning electron microscopy was applied to characterize the morphology of the silver microparticles on the nichrome surface. The micron-silver nichrome electrode exhibits a sensitive electrochemical response to H2O2 in pH 4.5 buffer solution, and its catalytic activity is much higher than that of a pure silver electrode. Under the optimum conditions, the well-defined reduction peak current increases linearly with H2O2 concentration in the range of 9.8 × 10−5∼5.9 × 10−3 M, and the detection limit is 0.58 μM. The electrode can be applied to the voltammetric determination of H2O2, and recoveries of artificial samples are between 99.7%–107.0%.

Graphene, a novel class of carbon nanostructure, has great promise for use as a sorbent material because of its ultrahigh specific surface area. A new method using a column packed with graphene as sorbent is developed for the preconcentration of trace amounts of chromium(III) prior to its determination by flame atomic absorption spectrometry. Some effective parameters on the extraction and complex formation were selected and optimized. The optimum experimental conditions of the proposed method were: pH, 8.0; amount of chelating agent, 2.0 mL of 0.1 mol L−1 8-hydroxyquinoline solution; eluent type and its volume, 2.0 mL of 2.0 mol L−1 nitric acid; flow rates of sample and eluent solution, 2.0 mL min−1. Under optimum conditions, an enrichment factor of 125 was obtained. The calibration graph was linear in the concentration range of 10.0–1000.0 μg L−1 with a correlation coefficient of 0.9995. The detection and quantification limits were 0.5 and 1.6 μg L−1, respectively. The relative standard deviation for ten replicate measurements were 4.3% for 20.0 μg L−1 and 3.4% for 800.0 μg L−1 of Cr(III). The proposed method was applied to tap water, sea water, and river water, and its accuracy was assessed through the analysis of certified reference water and recovery experiments. The recoveries of spiked samples were in the range of 95.7–101.2%.

Spectrofluorometry determination of chelerythrine by TAIL-DLPME: ionic liquid serves as both extractant and solvent by Li-bing Zhang; Hao Wu; Li-ming Du; Guang-quan Wang; Yin-xia Chang (1117-1121).
In the present study, a new ionic liquid extraction method was developed for the sensitive and accurate determination of chelerythrine in an aqueous solution. The results show that chelerythrine fluorescence increases significantly in ionic liquids, with 1-hexyl-3-methy-limidazolium hexafluorophosphate ([C6MIM][PF6]) having the greatest increase. Based on the significant enhancement of extraction efficiency of chelerythrine in ionic liquids, a sensitive spectrofluorometry method for the quantitative estimation of chelerythrine was established. At optimum conditions, a linear relationship was obtained in the range of 0.2–130 ng mL−1. The detection limit was 0.059 ng mL−1. The enrichment factor was 81.7, and the extraction recovery for chelerythrine was 98.1%. The proposed method was successfully applied for the determination of the chelerythrine in urine and serum samples.

Volatile species generation-flameless/flame atomization-atomic fluorescence spectrometry (VSG-FL/FA-AFS) without chromatographic separation was developed for quantitative determination of inorganic mercury (IHg) and methylmercury (MeHg) in water and biological samples by using a commercially available atomic fluorescence spectrometer. In the proposed method, inorganic mercury was measured by using the flameless mode. However, by using the flame atomization mode, the volatile hydride of MeHg generated by KBH4 can be transformed to elemental mercury vapour in the reductive argon–hydrogen flame, and both MeHg and IHg give a fluorescence signal in this flame atomization mode. Then, the concentration of MeHg can be calculated by subtracting IHg concentration from the total mercury concentration. The limits of detection for IHg and MeHg were 0.03 μg L−1 and 0.05 μg L−1, respectively. Spiked environmental water samples including seawater, tap water and river water were determined and satisfactory recoveries were obtained for MeHg and IHg. Furthermore, the proposed method was successfully applied to analyze biological certified reference material, TORT-2 (lobster hepatopancreas).

The dynamic nonlinear response of a semiconductor gas sensor was investigated to enhance the selectivity of the sensor response toward sample gases. A cyclic temperature composed of a fundamental harmonic was applied to a SnO2 semiconductor gas sensor and the resulting conductance of the sensor was evaluated by fast Fourier transformation (FFT). The dynamic nonlinear responses to samples (hydrocarbons and alcohols) were further characterized depending on the range and frequency of the cyclic temperature. These characteristic sensor responses were considered theoretically based on a reaction–diffusion model for the semiconductor surface.

A fast gas chromatographic method for simultaneous determination of 50 organochlorine (OCP) and pyrethroid (PP) pesticides in Lycium barbarum L. is established. OCP and PP were extracted from L. barbarum L. using acetonitrile, cleaned with alumina neutral-florisil column, and eluted by mixed solvents of ethyl acetate and hexane (15 : 85, v/v). Selected pesticides were identified using HP-5 and DB1701 capillary dual column and detected by electron-capture detector. Quantitative analysis was performed using an external standard by HP-5 capillary column. Results show that recoveries were 70.3%–115.7%, and the relative standard deviations (RSDs) were 2.0%–14.4%. The limits of detection of the method were 0.0023–0.0075 mg kg−1, and the limits of quantity were 0.0076–0.0247 mg kg−1. The method can treat multiple samples simultaneously. The results are reproducible and satisfactory.

In this study the analytical performance of three QuEChERS-based sample preparation procedures for multi-residue pesticide analysis were evaluated on four different vegetable oil matrices (olive oil, sunflower oil, palm oil and rapeseed oil) by LC-MS/MS. Different combinations of PSA (primary secondary amine), C18 and GCB (graphitized carbon black) sorbents were studied for the selected matrices in an effort to find out the most suitable clean-up approach for the selected types of vegetable oils. The effects of the different level on clean-up were evaluated by monitoring the method performance main parameters (recovery values, matrix effects and limits of detection) for a group of 44 relevant pesticides important in the cultivation of oil plants. From the aspect of matrix effects and LOD studies, the PSA + C18 + GCB sorbent-combination was the most suitable for the analysis of vegetable oils, since this method provided the lowest matrix effects and good LODs. However, when this method was applied, poor recovery was achieved (only 16% of the recovery results were acceptable), as the GCB sorbent adsorbed a remarkable part of the analytes. When GCB was omitted, good recoveries were obtained from all the four vegetable oil matrices tested (91% of results were acceptable) with good reproducibility, indicating that the effectiveness of the clean-up step should be sacrificed to obtain adequate recoveries. The further exclusion of C18 sorbent remarkably decreased the method performance in the case of palm oil and rapeseed oil, indicating that the PSA + C18 combination should be applied, while the applied quantity of GCB sorbent should be carefully adjusted.

Determination of quinocetone and its two major metabolites in chicken liver and muscle tissues by liquid chromatography-tandem mass spectrometry by Junjian Fang; Yong Li; Shengming Wu; Kunpeng Ma; Haijing Li; Zhixian Gao; Fangting Dong (1149-1154).
A sensitive and quantitative liquid chromatography-tandem mass spectrometry method for the simultaneous determination of quinocetone (QTN), a new antimicrobial growth agent, and its two major metabolites de-monoxy-quinocetone (DMO-QTN) and de-dioxy-quinocetone (DDI-QTN) in chicken liver and muscle tissues was developed and validated. Analyte extraction from samples is effectively performed using liquid–liquid extraction by methanol. The assays were based on reversed phase liquid chromatography (LC) coupled with tandem mass spectrometry in the electrospray, positive ion mode using selected reaction monitoring for analyte quantification. Calibration curves showed good linearity for all compounds in the concentration range of 2–500 μg kg−1. The detection limits for measured compounds were all ≤0.003 μg g−1 and quantification limits were all ≤0.010 μg g−1. Intra- and inter-assay CVs were all <10%. The recoveries ranged from 95% to 108%. Finally, the method was applied to real chicken liver and muscle samples, the quinocetone and its two metabolite residues were found only in liver.

A nano-nickel electrochemical sensor for sensitive determination of chemical oxygen demand by Tao Jing; Yusun Zhou; Qiaolin Hao; Yikai Zhou; Surong Mei (1155-1159).
Nano-nickel particles (nano-Ni) were electrodeposited on the surface of a glassy carbon (GC) electrode under −0.9 V for 25 min, and then used to construct an electrochemical sensor for sensitive determination of chemical oxygen demand (COD). The parameters such as electrolyte, deposition potential, deposition time and surface morphology were investigated. It was shown that nano-Ni film with high electrocatalytic activity was stably modified on the surface of the GC electrode, which could be used to effectively oxidize a wide spectrum of organic compounds. Under optimized conditions, the linear range was 10–1533 mg L−1 and the detection limit was as low as 1.1 mg L−1. Subsequently, this sensor was used to detect the COD values of different water samples and the results were linearly correlated to those by the classic dichromate method (R = 0.9493, p < 0.01, n = 54). This confirmed the reliability and practicality of the proposed method in the determination of COD in various water samples.

A novel pre-separation technique for the determination of divalent trace heavy metals in a soil sample was developed, in which deferoxamine was used as a masking agent for trivalent metal ions, Al3+ and Fe3+, and a chelate column was applied to collect target heavy metal ions and to eliminate alkali metal and alkaline earth metal ions. Deferoxamine had such a strong and selective masking effect on Al3+ and Fe3+ that divalent trace heavy metals in the soil sample were well separated from considerably greater amounts of Al3+ and Fe3+. Consequently, the determination of four trace metals (Ni, Cu, Zn, and Pb) in a certified soil reference material (JSAC-0401) was successfully performed using electrospray ionization mass spectrometry (ESIMS).

Separation of β-agonists in pork on a weak cation exchange column by HPLC with fluorescence detection by Wenying Meng; Jie Wei; Xi Luo; Feifang Zhang; Bingcheng Yang; Zhimou Guo; Xinmiao Liang (1163-1167).
The separation of β-agonists was performed for the first time on a silica-based weak cation exchange (WCX) stationary phase in which aqueous electrolyte solution with small amount of organic solvent was used as the mobile phase. Compared to the organic solvent used in reverse phase HPLC mode, the merits of using an aqueous mobile phase include unique selectivity, environment-friendly operation and reduced operation costs. The effective separation of four commonly used β-agonists including salbutamol, terbutaline, metoprolol and ractopamine, were achieved with the mobile phase of 20 mM ammonium formate/acetonitrile (5%). The interference from the hydrophobic analytes could be easily eliminated due to their poor retention under this mode. The detection limits for the above analytes when fluorescence detection is employed were in the range of 3.55–8.32 μg kg−1 and the spiked recoveries for metoprolol for the spike sample reached about 83% and the relative standard deviation (RSD) was 4.7%.

Diffusion-edited NMR spectra of heparin contaminants by John F. K. Limtiaco; Christopher J. Jones; Cynthia K. Larive (1168-1172).
Diffusion NMR is a useful method for the analysis of mixtures, providing information about the number of components as well as insight into their relative sizes. This work explores the use of diffusion-edited NMR measurements for the identification of the glycosaminoglycan impurities dermatan sulfate (DS), chondroitin sulfate (CS), and oversulfated chondroitin sulfate (OSCS) in solutions of heparin subjected to enzymatic digestion. Enzymatic digestion significantly improves the resolution of the biopolymer impurities in diffusion-edited 1H NMR spectra facilitating identification of high molecular weight contaminants.

A new simple, sensitive and cost effective supramolecular-based dispersive liquid–liquid microextraction (SM–DLLME) has been reported in very high concentration of salt. The contaminant used as a model was Orange II, an anionic dye, and was preconcentrated without any derivatization or ion-pair formation reaction. This new method can determine the analytes in high concentration of salt (up to 5 M) and does not need any kind of disperser agent. In the novel technique developed here, decanoic acid (extraction solvent) is dissolved in a mixture of tetrabutylammonium hydroxide (Bu4NOH) (self-assembly agent) and tetrabutylammonium bromide (Bu4NBr) (salting-in agent) which make up the vesicles and extract the analytes from samples. Several important factors influencing the extraction efficiency of Orange II and its subsequent determination were investigated and optimized. Under the optimized conditions and preconcentration of only 5.00 ml of sample, the enhancement factor was 74, limit of detection (LOD) was 0.35 μg L−1 and relative standard deviations (RSDs) for 45 μg L−1 and 100 μg L−1 of Orange II in different samples were 3.7% and 2.9%, respectively (n = 6).

Introduction of nanogold–DAB as a HRP substrate for simplifying detection in visual DNA microarrays by Hong-Juan Qi; Su-Hong Chen; Rong-Zhang Hao; Hua Shi; Min-Li Zhang; Sheng-Qi Wang (1178-1181).
Tyramine signal amplification, coupled with a gold-label silver stain (TSA–GLSS), has been demonstrated to be a sensitive visual assay for DNA microarrays. However, the procedure is laborious and time-consuming as nanogold particles do not accumulate directly in the vicinity of horseradish peroxidase (HRP). Instead, nanogold particles must accumulate through two procedures, including biotin-tyramine deposition via HRP catalysis and specific binding of biotin and streptavidin, followed by the introduction of streptavidin-nanogold. These conditions restrict this method's utility in DNA microarray detection. In the present study, nanogold was covalently linked to 3,3′-diaminobenzidine (DAB) to form a conjugate for the detection of Salmonella typhi. DAB can be oxidized by H2O2 and thus accumulate nanogold–DAB via HRP catalysis, leading to direct nanogold deposition. The results indicated that the detection limit of nanogold–DAB assay reached 103 CFU mL−1, which was comparable with that of TSA–GLSS. As one incubation step was omitted and the silver-staining time greatly shortened, this nanogold–DAB assay was more convenient than TSA–GLSS and offers great potential in detecting low concentrations of target DNA.

Back matter (1182-1182).

Back cover (1183-1184).