Analytical Methods (v.7, #14)

Front cover (5717-5718).

Contents list (5719-5730).

Advanced detection methods for traceability of origin and authenticity of olive oils by Gaozhi Ou; Rui Hu; Liangxiao Zhang; Peiwu Li; Xinjian Luo; Zhaowei Zhang (5731-5739).
The adulteration of olive oils based on advanced sensors has attracted high interest owing to its health benefits in the prevention and treatment of certain pathologies. Concerning its health and commercial aspects, lower grade oil blending and other illegal additives in virgin olive oil can negatively affect the nutritive value of olive oil. This review focuses on the advances in the sensing and identification of adulteration of olive oil. Optical sensing, chromatography (usually coupled with mass spectrometry), and nuclear magnetic resonance are discussed in detail. Other methods including a DNA-based method, dielectric spectroscopy, differential scanning calorimetry, thermogravimetric analysis and electronic nose among others, are overviewed as well.

Screening and authentication of herbal formulations based on microextraction-assisted voltammetry of microparticles by Mariele Martini; Leandro Machado de Carvalho; Adela Blasco-Blasco; Antonio Doménech-Carbó (5740-5747).
A simple solid state electrochemical methodology for screening and authentication of herbal formulations is described. The proposed method is based on the recording of the voltammetric response, in contact with aqueous buffers, of microparticulate films of antioxidant compounds resulting from micro-extraction of dried herbal samples with ethanol or acetone. The obtained voltammetric responses led us to differentiate between diverse active components upon application of bivariate and multivariate chemometric techniques. Resolution of herbal preparations containing two or more components is possible when well-separated voltammetric signals are recorded. In favorable cases, such characteristic voltammetric signatures can be used for relative quantification of components and synthetic adulterants.

A selective and robust methodology for the analysis of nine N-nitrosamines (NAs), N-nitrosodimethylamine (NDMA), N-nitrosomorpholine (NMor), N-nitrosomethylethylamine (NMEA), N-nitrosopyrrolidine (NPyr), N-nitrosodiethylamine (NDEA), N-nitrosopiperidine (NPip), N-nitroso-n-dipropylamine (NDPA), N-nitrosodi-n-butylamine (NDBA) and N-nitrosodi-n-phenylamine (NDPhA), was developed and validated. This method is based on ultra-high-performance liquid chromatography (UHPLC) coupled to mass spectrometry using heated electrospray ionization (HESI) in positive ionization mode with a Q-Exactive mass spectrometer. After the selection of a suitable column for NA separation, the mobile phase and the injection volume as chromatography parameters were optimized. Mass spectrometry operating parameters, including sheath gas, auxiliary gas, spray voltage, S-Lens RF Level, resolution, automatic gain control (AGC) target and maximum injection time, were also optimized in order to maximize the instrument analytical signal response. The method was optimized and validated in HPLC grade water, drinking water and wastewater matrices with satisfactory results. For accurate quantification, NDMA-d6 and NDPA-d14 were used as internal standards. The extraction recoveries in real matrices ranged from 68–83% for eight of the nine target nitrosamines, except for NDPhA with values of 22–31%. The detection limits ranged from 0.4 to 12 ng L−1. Analytical results revealed a trace concentration of NDPhA (1.2 ng L−1) in one of the analyzed water matrices. This work demonstrates that nitrosamines can be analyzed using LC-MS, on a Q-Exactive instrument, offering a faster alternative to the traditional GC-MS methods. The use of high resolution accurate mass spectrometry helps to obtain good selectivity for the detection of both GC-detectable and GC-undetectable compounds.

Although patterning hundreds or thousands of electrochemical electrodes on lab-on-a-chip devices is straightforward and cost-effective using photolithography, easily making connections between hundreds of electrodes and external amplifiers remains a bottleneck. Here we describe two electrode addressing approaches using multiple fluid compartments that can potentially reduce the number of external connections by ∼100-fold. The first approach enables all compartments on the device to be filled with solution at the same time, and then each fluid compartment is sequentially electrically activated to make the measurements. The second approach achieves lower measurement noise by sequentially filling recording chambers with solution. We propose an equivalent circuit to explain measurement noise in these recording configurations and demonstrate application of the approaches to measure quantal exocytosis from individual cells. A principle advantage of using these approaches is that they reduce the fraction of the microchip area that needs to be dedicated to making external connections and therefore reduces the cost per working electrode.

ICP-MS DNA assay based on lanthanide labels and hybridization chain reaction amplification by Chong Deng; Chong-Hua Zhang; Hao Tang; Jian-Hui Jiang (5767-5771).
A novel ICP MS DNA assay based on lanthanide labels and hybridization chain reaction amplification has been developed. This approach utilizes an enzyme-free amplification and can be operated under mild conditions. The developed assay exhibits desirable sensitivity and is suitable for multiple DNA target analysis because a general DNA hybridization approach is applied to capture target DNA. Considering lanthanide labels and ICP-MS have excellent ability in high-level multiplexing analysis, this approach holds great potential for practical use in quantitative determination of DNA targets for future clinical applications.

Determination of flumetsulam residues in 20 kinds of plant-derived foods by ultra-performance liquid chromatography coupled with tandem mass spectrometry by Pingping Wang; Fajun Tian; Jun Xu; Fengshou Dong; Shasha Li; Yongquan Zheng; Xingang Liu (5772-5779).
A modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method for the simultaneous quantitative determination of flumetsulam in 20 kinds of complicated plant-derived food grains (rice, wheat, and corn), vegetables (cabbage, celery, tomato, kidney bean, eggplant, potato, radish and leek), fruits (apple, peach, grape and orange), edible mushroom (mushroom), tea (green tea), nuts (almond), oil crops (soybean) and edible oil (peanut oil) was developed and validated using ultra-performance LC coupled with MS/MS. Acetonitrile was the extraction solvent and simultaneous liquid–liquid partitioning was formed by adding anhydrous magnesium sulfate (MgSO4) and sodium chloride (NaCl). The extract was then cleaned up by dispersive-SPE using graphitized carbon black (GCB), almond and peanut oil using Cleanert Florisil, as selective sorbents. The determination of the target compound was achieved in less than 6.0 min using an electrospray ionization source in positive mode (ESI+). Quantification was performed by calibration curves of standards from 0.01–1.0 mg L−1 with r2 = 0.999. Further optimization of sample preparation and determination achieved recoveries between 73.85% and 115.76% for all analytes with RSD values lower than 10% in 20 kinds of plant-derived foods at three levels (10, 50 and 100 μg kg−1).

A wavelength selection method for spectroscopic analysis, named correlation coefficient optimization coupled with partial least-squares (CCO-PLS), is proposed, and was successfully employed for reagent-free ATR-FTIR spectroscopic analysis of albumin (ALB) and globulin (GLB) in human serum. By varying the upper bound of correlation coefficient between absorbance and analyte's content, the CCO-PLS method achieved multi-band selection. Two PLS-based methods, which used a waveband having positive peaks of the first loading vector (FLV) and a combination of positive peaks of the correlation coefficient spectrum, were also conducted for comparison. Based on the leave-one-out cross-validation for CCO-PLS, appropriate waveband combinations for ALB and GLB were selected, the root-mean-square errors of prediction for validation samples were 1.36 and 1.35 (g L−1) for ALB and GLB, respectively, which were better than the two comparison methods. The CCO-PLS method provided a new approach for multi-band selection to achieve high analytical accuracy for molecular absorption bands that were composed of several spaced wavebands.

Water-soluble and dual-emitting bovine serum albumin-templated bimetallic platinum–gold fluorescent nanoclusters (BSA–Pt–Au NCs) have been synthesized for the first time by a simple and rapid one-pot synthesis strategy at a vital molar ratio of Pt/Au precursors under basic conditions. The BSA–Pt–Au NCs show unique and well-resolved dual-emission bands at 405 nm and 640 nm corresponding to the emission of the BSA–metal complex and Au NCs under the excitation at 340 nm, respectively. Interestingly, with the addition of mercury(ii) (Hg2+) ions, the emission intensity at 405 nm (F405) has a negligible influence, while the emission intensity at 640 nm (F640) decreases dramatically, which is due to the specific and strong d10–d10 interactions between Hg2+ ions and Au atoms/ions on the surface of the BSA–Pt–Au NCs. Meanwhile, cysteine (Cys) could effectively block Hg2+ ions to quench the fluorescence of BSA–Pt–Au NCs, which is based on the strong interaction between Hg2+ ions and the thiol functional group. These allowed the analysis of Hg2+ ions and Cys with the ratio of two emission intensities (F405/F640) in ultra-sensitivity and selectivity with high accuracy. The limit of detection (LOD) was 0.3 nM for Hg2+ ions and 0.04 μM for Cys (S/N = 3), respectively. This work indicates that the fluorescent sensor possesses potential applications for biological detection.

Novel fluorescent, molecularly imprinted polymers (MIPs) were synthesized to detect sulfadiazine (SDz), an antibiotic used in animals that produce food for human consumption. Radical polymerization between a novel fluorescent monomer 7-acryloxy-4-methylcoumarin, ethylene glycol dimethacrylate and methacrylic acid, in the presence of SDz as a molecular template, afforded the target MIPs. The photoluminescence properties of these MIPs were studied and found to exhibit stable, enhanced fluorescence emission. The fluorescence emission of both the MIPs and non-imprinted polymers (NIPs) was quenched in the presence of SDz and similar analogues. Significantly, the MIPs were more sensitive than the NIPs for the SDz analyte, confirming that molecular imprinting imparted selectivity. The fluorescence quenching ratio of the MIPs exhibited a linear decrease with increasing concentrations of SDz in the range of 1.0–40 μmol L−1 with a detection limit of 0.48 μmol L−1. Importantly, the MIPs proved successful in determining SDz concentrations in spiked milk samples and showed superior recovery from 85.73% to 101.37%.

A procedure involving QuEChERS sample extraction combined with dispersive liquid–liquid microextraction (DLLME) and stacking as off-line and on-line preconcentration techniques followed by micellar electrokinetic chromatography (MEKC) has been developed for the determination of five organophosphorus pesticides (dimethoate, phosphamidon, paraoxon-methyl, paraoxon and fensulfothion). The important parameters that influence the stacking and DLLME efficiency were evaluated. The RSDs of the migration time ranged from 0.06% to 1.03% and the peak area ranged from 1.12% to 5.15% for the five analytes, indicating the good repeatability of the method. The method was extensively validated by evaluating the linearity (r2≥ 0.9956), LODs (0.010–0.018 μg mL−1) and recovery (78.75–118.15%). The QuEChERS–DLLME–stacking–MEKC method has been successfully applied to assay the five organophosphorus pesticides in Astragalus membranaceus. Under the optimized conditions, the proposed method provided a 90.0- to 167.3-fold enrichment of the five pesticides compared with the normal MEKC method, which offers an ideal solution for the determination of some trace pesticides in real samples with complex matrices.

Accessible glyco-tripod amphiphiles for membrane protein analysis by Manabendra Das; Hyoung Eun Bae; Ho Jin Lee; Juyeon Go; Pil Seok Chae (5808-5813).
Membrane protein manipulation is known to be an extremely challenging task, mainly because of incompatibility between the hydrophobic surface area of proteins and the hydrophilic character of an aqueous medium. To avoid protein degradation resulting from this incompatibility, detergents are used as essential tools in the study of membrane proteins. However, traditional detergents have a limited ability to stabilize the native conformation of membrane proteins. This study introduces a novel tripod amphiphile that can be prepared efficiently from a commercially available compound. The new agent proved effective for the long-term stability of a multi-subunit superassembly, a membrane protein sensitive to denaturation.

Sensitive and fluorescence “turn-on” detection of BRCA1 and TB4 DNA sequences using water-soluble conjugated polythiophenes by Yun Ma; Yong Xia; Liqiang Yan; Fang Wang; Zhihui Miao; Meifang Cui; Hongtao Yao; Zhengjian Qi (5814-5819).
A biocompatible, label-free and sensitive fluorescence “turn-on” approach was designed to detect BRCA1 and TB4 DNA sequences using poly(3-[(S)-5-amino-5-carboxyl-3-oxapentyl]-2,5-thiophenylene hydrochloride) (POWT). The fluorescence intensity of POWT in solution could be efficiently quenched by adding either BRCA1 or TB4 DNA sequences. The Stern–Volmer equation was used to fit the quenching effect and the LOD could thus be obtained. The fluorescence would recover its initial intensity after the addition of completely complementary ssDNA, but not with sequences with 1–3 mismatched bases. The influences of buffer pH and buffer composition were investigated systematically in order to improve the sensitivity of this fluorescent probe. For the BRCA1 DNA sequence, the KSV (Stern–Volmer constant) and LOD were 8.254 × 109 M−1 and 2.231 × 10−12 M, respectively. For the TB4 DNA sequence, the KSV and LOD were 6.924 × 109 M−1 and 2.659 × 10−12 M, respectively. In addition, the quantitative linearity range for detecting these two DNA sequences was from 1 × 10−11 M to 5 × 10−11 M, and the response time for the quenching effect was less than 10 minutes for both sequences. Hence, with fine biocompatibility, high sensitivity, fast response and high recovery rate, a serine functionalized POWT might be a promising candidate for detecting the BRCA1 DNA sequence and TB4 DNA sequence as well as their mutations, which might possibly be applied to clinical diagnosis of breast cancer or ovarian tumours.

A rapid, convenient, sensitive and reliable dispersive liquid–liquid microextraction (DLLME) method was coupled with field-amplified sample injection (FASI) in capillary electrophoresis with a diode array detector for the quantification of verapamil enantiomers in human plasma samples. Various parameters affecting the extraction efficiency as well as FASI were optimized. The method performance was studied over the concentration range of 25–350 ng mL−1 for each enantiomer in terms of accuracy (recovery = 92–115%), linearity (coefficients of determination (R2) > 0.99) and repeatability (RSDs% agree within 15%). The method was validated in plasma according to FDA guidelines. This is the first work showing the possibility of the use of DLLME and on-line sample pre-concentration techniques for the analysis of verapamil enantiomers in plasma.

Method validation for the analysis of licorice acid in the blending process by near infrared diffuse reflectance spectroscopy by Zhong Xue; Bing Xu; Chan Yang; Xianglong Cui; Jianyu Li; Xinyuan Shi; Yanjiang Qiao (5830-5837).
The present work describes the validation of the near infrared (NIR) method for the quantification of the concentration of licorice acid in the blending process of licorice and talcum powder mixtures. The NIR diffuse reflectance spectra of samples were collected during the mixing process and the partial least squares (PLS) model was developed. The accuracy profile (AP) approach that was fully compliant with the ICH Q2 (R1) guidelines was used in order to assess the validity of the NIR chemometric method. Particularly, the β-content, γ-confidence tolerance interval, instead of the β-expectation tolerance interval in the AP methodology, was introduced to provide a better estimate of measurement risk. The quantitative validation criteria such as trueness, precision (both repeatability and intermediate precision), result accuracy and valid range were obtained. The lower limit of quantification (LLOQ) was 1.26 mg g−1. The results demonstrated that NIR spectroscopy is suitable for the analysis of the concentration of licorice acid. And the risk of using the established analytical method in the routine phase could be well visualized and controlled.

A novel fluoroquinolone molecularly imprinted polymer (MIP) on the surface of ethylenediamine-functionalized magnetic carbon nanotubes (EDA@Mag-CNTs–MIP) was synthesised. The properties of the EDA@Mag-CNTs–MIP were characterized by using a transmission electron microscope (TEM) and vibrating sample magnetometer (VSM). With using EDA@Mag-CNTs–MIP as the adsorbent, a simple and sensitive method based on the use of a magnetic molecularly imprinted polymer matrix solid phase dispersion extraction (Mag-MIP-MSPD) procedure combined with ultra-fast liquid chromatography-tandem quadrupole mass spectrometry (UFLC-MS/MS) was developed to determine 12 fluoroquinolones in river water. Fluoroquinolones adsorbed with EDA@Mag-CNTs–MIP could be simply and rapidly isolated through a magnetic field, and the main factors, i.e., the pH value and sample volume, affecting the extraction were investigated in detail. Under optimal experimental conditions, the enhancement factor of the Mag-MIP-MSPD procedure was 100 times. The obtained results demonstrated the higher extraction capacity of EDA@Mag-CNTs–MIP with recoveries between 80.2 and 116%. The limits of quantification (LOQs) for the fluoroquinolones were between 0.26 and 1.78 ng L−1. The developed Mag-MIP-MSPD UFLC-MS/MS method had been successfully applied to sixty real samples, and it was confirmed that the EDA@Mag-CNTs–MIP was a kind of highly effective Mag-MIP-MSPD material for fluoroquinolone analyses.

5-(p-Dimethylaminobenzylidene) rhodanine-modified magnetic halloysite nanotubes as a new solid phase sorbent for silver ions by Mohammad Amjadi; Azam Samadi; Jamshid L. Manzoori; Naser Arsalani (5847-5853).
In this work, magnetic halloysite nanotubes (MHNTs) were modified with 5-(p-dimethylaminobenzylidene) rhodanine by using a facile procedure and the modified MHNTs were used as a new and inexpensive magnetic solid phase extraction sorbent for the separation and preconcentration of Ag(i) ions from environmental samples. The factors affecting the adsorption of μg L−1 levels of silver onto the sorbent were investigated. Quantitative recoveries (>95%) were obtained for acidic solution (pH = 3) using 0.05 g of sorbent. Under the optimum conditions, the calibration graph was linear in the 4.0–200 μg L−1 concentration range, with a detection limit of 1.6 μg L−1. The selectivity of the method was studied and no serious interference was observed. This method was applied for the determination of trace silver in various water and soil samples.

Fluorescence polarization immunoassays for carbamazepine – comparison of tracers and formats by Lidia Oberleitner; Sergei A. Eremin; Andreas Lehmann; Leif-Alexander Garbe; Rudolf J. Schneider (5854-5861).
For the antiepileptic drug and anthropogenic marker carbamazepine (CBZ), a fast and cost-effective immunoassay based on fluorescence polarization (FPIA) was developed. The required fluorophore conjugates were synthesized from different fluorescein and CBZ derivatives. The most suitable tracer was CBZ–triglycine–5-(aminoacetamido)fluorescein. Additionally, the applicability of the assay in tubes and on microtiter plates was tested. The first format can be performed in a portable instrument and therefore can be applied in field measurements. The measurement of an individual sample can be carried out within 4 min. This assay shows a measurement range of 2.5–1000 μg L−1 and a test midpoint (or IC50) of 36 μg L−1. The FPIA performed on microtiter plates is useful for the assay development and is suitable for a very high throughput (up to 24 samples in 20 min). The test midpoint of this assay is 13 μg L−1 and the measurement range is 1.5–300 μg L−1. Furthermore, this assay requires smaller sample volumes and less reagents, including the crucial amount of antibody. The applicability of both assays to spiked surface water samples was evaluated. The recovery rates vary between 66–110% on microtiter plates and 81–140% in tubes.

The residual glyphosate (GLYP) in the environment is harmful to human health. Monitoring GLYP and its major metabolite aminomethylphosphonic acid (AMPA) in a complicated matrix is an important analytical task. Based on the affinity of phosphate groups to metal ions, iron oxide nanoparticles immobilized with Ti4+ using polydopamine (PDA) as bridging molecules (Fe3O4@PDA-Ti4+ NPs) were employed to develop a magnetic solid phase extraction (mSPE) method for GLYP and AMPA by electrostatic interaction. After extraction with Fe3O4@PDA-Ti4+ NPs and elution for 5 min, the purified and enriched analytes were derivatized, prior to CE with diode array UV detection. Relying on the coupled mSPE-CE method, the detection limits were 0.4 ng mL−1 for GLYP and AMPA. The present method was employed to monitor GLYP and AMPA in a Roundup® sample and river water sample, revealing its good possibilities for applicability. Herein, by virtue of the immobilized metal affinity extraction (IMAE), the coupled mSPE-CE method would be effective, easy to operate and applicable for analyzing herbicides in environmental samples.

A novel strategy for the classification of naturally colored cotton fibers based on digital imaging and pattern recognition techniques by Maria Ivanda S. Gonçalves; Welma T. S. Vilar; Everaldo Paulo Medeiros; Márcio José Coelho Pontes (5869-5875).
This study proposes the use of digital images and multivariate analysis as an alternative methodology for the classification of naturally colored cotton fiber, according to cultivar type. For this purpose, two groups were evaluated: the first comprised of BRS 200 Marrom and BRS Topázio cultivars, while the second contained BRS Rubi and BRS Safira cultivars. Cotton fiber sample images were obtained using a webcam and the frequency distribution of color indexes on the grayscale, red–green–blue (RGB), hue (H), saturation (S), value (V), and grayscale channels was obtained. Classification models based on linear discriminant analysis (LDA) with prior variable selection by successive projection algorithm (SPA) and stepwise (SW) were used. For the purpose of comparison, partial least squares discriminant analysis (PLS-DA) applied to the full-histogram was also used. For both groups, the best results were achieved using the LDA/SW model, with a correct classification rate (CCR) of 96% for the prediction set using the HSV combination. The proposed method is simple, low cost, does not use a reagent, does not destroy the sample and provides results in a short period of time.

Electrochemical response of agar ionogels towards glucose detection by Anshu Sharma; Kamla Rawat; Pratima R. Solanki; H. B. Bohidar (5876-5885).
We have reported a sensing platform comprising of agar ionogels (IGs) made in ionic liquid solutions (1-octyl-3-methyl imidazolium chloride [C8mim][Cl] and 1-ethyl-3-methylimidazolium chloride [C2mim][Cl]) and used it for glucose oxidase (GOx) immobilization for glucose detection. The ionogels were deposited onto an indium tin oxide (ITO) coated glass plate using the drop-casting technique. These agar–[C8mim][Cl]/ITO (Ag–C8/ITO) and agar–[C2mim][Cl]/ITO (Ag–C2/ITO) substrates were used for immobilization of GOx, which was selected as a model enzyme to investigate its interaction with these electrodes using electrochemical techniques. Structural and morphological studies of these GOx/Ag–C8/ITO and GOx/Ag–C2/ITO electrodes were performed by using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and electrochemical techniques (CV). These GOx/Ag–C8/ITO and GOx/Ag–C2/ITO bioelectrodes exhibited improved glucose detection capability in the concentration range of 0.27–16.7 mM and 0.28–5.6 mM with sensitivity ≈4.1 μA mM−1 cm−2 and 14.6 μA mM−1 cm−2, respectively. The values of apparent Michaelis–Menten constant (Km) were 0.023 mM and 0.0007 mM for the aforesaid two cases respectively. It is shown that customization of agar hydrogels in green solvent medium widens the scope of their potential applications in glucose sensing in real samples.

Wooden-tip electrospray ionization mass spectrometry for trace analysis of toxic and hazardous compounds in food samples by Bi-cheng Yang; Feng Wang; Wei Deng; Yang Zou; Fa-ying Liu; Xi-di Wan; Xiao Yang; Huai Liu; Ou-ping Huang (5886-5890).
Wooden-tip electrospray ionization (wooden-tip ESI) is applied to the rapid, in situ, direct qualitative and quantitative trace analysis of toxic and hazardous compounds in food samples. To evaluate the potential of wooden-tip ESI mass spectrometry (MS) in food analysis, pesticides, toxicants, date-rape drugs, and illicit additives were detected in various food samples. Wooden-tip ESI-MS experiments were performed using wooden tip sampling and ionization, with examination using MS and tandem mass spectrometry (MS/MS). The results proved that wooden-tip ESI allows the detection and confirmation of traces of toxic and hazardous compounds in food. In addition, selected analytes in beverages were obtained at absolute levels as low as ∼10 pg. Quantitation of analytes in liquid and powder samples was also evaluated. Single sample analysis was completed within 2 min. The data obtained shows that the wooden-tip ESI-MS is a promising tool for rapid analysis of food samples.

Based on the analysis of specific volatile substances released during beef spoilage, the beef status can be predicted. During beef spoilage, ammonia, carbon dioxide, alcohols, esters, aldehydes, ketones, and other substances are released and can be detected by long-path FITR (Fourier transform infrared spectroscopy). The concentrations of these substances vary with storage time. We could distinguish beef at different stages of spoilage using FTIR spectrometry combined with chemometric methods. This study indicates that infrared spectroscopy can be used to monitor the spoilage status of beef.

A flow-through enzymatic microreactor for the rapid conversion of triacylglycerols (TAG) into fatty acid ethyl ester (FAEE) or fatty acid methyl ester (FAME) derivatives was developed. The microreactor was a porous silica monolith fabricated within a 320 μm ID fused silica capillary with lipase from Candida antarctica immobilized onto the large surface area of the monolith. The microreactor was used for the room temperature ethanolysis of TAG from edible oils including canola, sesame, soybean and refined-bleached-deodorized palm oil. GC/MS-NCI and GC/FID were used to prove the identification of the FAEE and FAME products. The microreactor completely transformed the starting oils into FAEEs or FAMEs, without the use of any reagents other than alcohol, in quantities suitable for GC analysis. The prototype microreactors were reusable >5 times with ethanol and 2 times with methanol. The FAEE products obtained using the microreactor were similar to those produced using commercial Novozyme 435 enzyme beads as well as by catalysis with ethanolic H2SO4.

An improved method with a simple and rapid solid phase extraction procedure for the simultaneous determination of nine common polybrominated diphenyl ethers (PBDEs) in human serum has been established using gas chromatography-negative chemical ionization mass spectrometry. Purification and lipid removal were carried out directly on a solid phase extraction column using water, concentrated sulfuric acid and 10% aqueous methanol, followed by elution with dichloromethane. The eluent was concentrated and then loaded on a short capillary column to detect PBDEs. The results of the spike recovery experiments in fetal bovine serum showed that the average recoveries of the target compounds were between 80.5% and 110.2%. The intraday relative standard deviations were between 1.2% and 9.8%, and the interday relative standard deviations were between 1.8% and 11.1%. The limits of detection for tribromo to nonabromo diphenyl ethers in fetal bovine serum were in the range of 0.208–0.653 ng L−1 and the limits of quantification were in the range of 0.653–1.000 ng L−1. The limit of quantification for BDE-209 was 10.038 ng L−1 and the correlation coefficients in the linear range were greater than 0.997. This method showed good accuracy and precision, and was suitable for the determination of PBDEs in umbilical cord blood samples.

This work presents a novel approach for the simultaneous determination of mixtures of magnolol and honokiol in herb and plasma samples by combining the sensitivity of molecular fluorescence and the selectivity of the second-order calibration method. The excitation–emission matrix fluorescence data were processed by applying the second-order calibration method based on parallel factor analysis (PARAFAC) and self-weighted alternating trilinear decomposition (SWATLD) algorithms. The results showed that the method could solve the problem of analyzing complex multi-component samples, using “mathematics separation” to replace some or enhance chemical separation. The recoveries from spiked herb samples were in the range of 93–104% for magnolol and 89–98% for honokiol, and those from spiked human plasma samples were in the range of 98–105% for magnolol and 94–96% for honokiol. In herb samples, the LOD values of magnolol were 0.51 μg ml−1 and 0.45 μg ml−1, and those of honokiol were 0.37 μg ml−1 and 0.24 μg ml−1, when using PARAFAC and SWATLD. For human plasma samples, the LOD values of magnolol were 0.93 μg ml−1 and 0.64 μg ml−1, and 0.79 μg ml−1 and 0.42 μg ml−1 for honokiol. The results demonstrated that this method had both high recovery and good precision for the determination of honokiol and magnolol. The proposed method avoided preconcentration and had a simple disposal process, so it considerably decreased the analysis time and the experimental costs. Thus, it can be considered as a green analytical procedure for magnolol and honokiol determination in herb and plasma samples.

Liquid chromatography-tandem mass spectrometry coupled with an ultrasonic-assisted extraction and solid phase extraction procedure has been investigated for the separation and detection of imidazolium, pyridinium, pyrrolidinium, ammonium and phosphonium-based ionic liquid (IL) cations in soil samples. First, the samples were extracted with acetonitrile–0.2 mol L−1 ammonium chloride solution (8 : 2, v/v; pH 3) using an ultrasonic assisted extraction procedure and then eluted on a Strata-X-CW solid-phase extraction column. The target compounds were effectively separated with an Agilent SB-C18 (1.88 μm, 3.0 mm × 100 mm) chromatographic column by gradient elution, in which acetonitrile–water (containing 0.1% formic acid) acted as the mobile phase. The IL cations were determined by electrospray positive ionization (ESI+) combined with multiple reaction monitoring (MRM) mode, and quantified by the matrix-matched external standard method. The limits of detection (LODs) ranged from 0.15 to 2.88 ng g−1 and the limits of quantification (LOQs) from 0.50 to 9.50 ng g−1. The average recoveries of the twenty-three IL cations in soil samples were in the range of 69.3–106.8%, with relative standard deviations of 2.17–12.1%. This assay showed greater sensitivity and accuracy, to meet the requirements of the determination of IL cations in soil.

A new calibration curve calculation method for absolute quantification of drug metabolizing enzymes in human liver microsomes by stable isotope dilution mass spectrometry by Huanhuan Wang; Haifeng Zhang; Jiabin Li; Junying Wei; Rui Zhai; Bo Peng; Hailing Qiao; Yangjun Zhang; Xiaohong Qian (5934-5941).
Accurate quantification of cytochrome P450 (CYP) enzymes and uridine 5-diphospho-glucuronosyltransferase (UGT) enzymes is essential for the reliable assessment of the safety of new drugs and individual medicines. Stable isotope dilution-multiple reaction monitoring mass spectrometry (SID-MRM MS) has been used for the determination of drug metabolizing enzymes in complex biological samples, in which a working curve is often established by adding a series of light peptides into aliquots of a blank sample matrix and stable isotope-labeled (SIS) peptides. But when multiple proteins are simultaneously quantified, a blank sample matrix devoid of targeted proteins is difficult to prepare. To solve the problem, a linear curve was established by adding a series of SIS peptides to an actual sample instead of a heterologous or an artificial sample as a matrix, and a new calibration curve calculation method was proposed to calculate the concentrations of endogenous peptides or proteins in a biological sample as follows: a linear curve was first plotted with peak area ratios (SIS peptides/endogenous peptides) on the y-axis and the corresponding concentrations on the x-axis, and then the concentrations of endogenous peptides in a biological sample could be accurately obtained according to our mathematical formula. Finally, a working curve was built with peak area ratios on the y-axis and the corresponding concentration ratios on the x-axis, and when the peak area ratio of a transition of a peptide in a biological sample was measured and substituted into the working curve, the corresponding concentration ratio could be obtained to calculate the concentration of peptides. Experimental results demonstrated that the established method was reliable and sensitive with a recovery of 97.0%, the limit of quantification (LOQ) lower than 20 fmol, a linear range from 5 fmol, 10 fmol or 20 fmol–1000 fmol for different peptides and the coefficient of variation lower than 10%. The established method was applied to the determination of 21 drug metabolizing enzymes in five human liver microsomal samples, and the results are in agreement with the reported data, which proves that this method can be applied to the determination of targeted proteins in biological samples.

Saponins and flavonoids are the main bioactive ingredients in Camellia oleifera Abel. In this study, the fragmentation pathways of triterpene saponins in Camellia oleifera were investigated and confirmed by electrospray ionization linear ion trap-orbitrap (LTQ-orbitrap), and a new strategy for rapid characterization of saponins by ultra-high-pressure liquid chromatography with LTQ-orbitrap mass spectrometry (UHPLC-LTQ-orbitrap-MSn) was developed. Based on the summarized fragmentation rules, 36 triterpene saponins from the testa of C. oleifera extracted by n-butanol were found, 23 of which were completely and tentatively characterized. Besides, 35 saponins were identified as novel saponins. Additionally, 8 flavonol-O-glycosides were separated from C. oleifera, with 7 being tentatively characterized but one unambiguously identified, and one of the 7 flavonol-O-glycosides is reported for the first time in genus Camellia. This study provides a systematic strategy for the identification of saponins by the UPLC-MSn method in genus Camellia and summarized essential data for phytochemical studies of C. oleifera.

Spectroscopic studies on naftazone and its metal complexes with analytical applications for quality control of tablets by Fawzia Ahmed Ibrahim; Nahed Mahmoud El-Enany; Rania Nabih El-Shaheny; Ibraam Emad Mikhail (5954-5961).
The spectroscopic characteristics of the haemostatic drug naftazone (NFZ) as well as its metal complexing ability were investigated spectrophotometrically in order to develop new, simple and sensitive spectrophotometric methods for its determination. The first method was based on measurement of the absorbance of the drug in NaOH (2 × 10−3 M) at 494 nm. The method showed excellent linearity over the concentration range of 0.5–10.0 μg mL−1. The second method was based on complex formation between NFZ and different metal ions including Cu2+, Ni2+, Co2+ and Zn2+ in borate buffer. The absorbances of the formed complexes were measured at 512, 506, 498 and 502 nm for Cu2+, Ni2+, Co2+ and Zn2+, respectively. Beer's law was obeyed within the concentration ranges of 2.0–14.0, 2.0–10.0, 2.0–14.0 and 2.0–19.0 μg mL−1 for Cu2+, Ni2+, Co2+ and Zn2+complexes, respectively. The molar ratios and stability constants of the metal complexes were calculated and a proposal of the reaction pathway was consequently postulated. The proposed methods were validated according to ICH guidelines and they were successfully applied for the determination of NFZ in its tablet dosage forms without the interference from excipients normally added to tablets. The results of the proposed methods were favorably compared with those obtained by the comparison method using Student's t-test and variance ratio F-test.

Identification and quantitative determination of carbohydrate molecules in Greek honey by employing 13C NMR spectroscopy by Aristea Kazalaki; Maria Misiak; Apostolos Spyros; Photis Dais (5962-5972).
A methodology based on 13C NMR spectroscopy was employed to detect and quantify fourteen mono-, di- and trisaccharide molecules in authentic Greek honey samples with no prior separation. Unambiguous assignment of 13C NMR chemical shifts has been achieved by means of two-dimensional NMR techniques using sugar model compounds. The quantitative 13C NMR method was rigorously validated (accuracy, linearity, range, limit of detection, etc.) using either single sugar molecules, or artificial mixtures of isoglucose (glucopyranose and fructose) and global mixtures of fourteen model compounds. Subsequent integration of appropriate signals in the 13C NMR spectra allowed the quantification of these compounds. The present methodology has been applied to authentic Greek honey samples and provided quantitative results for 28 sugar tautomers. The observed differentials in the content of these biomarkers amongst the various honey samples of different botanical origins are expected to form the basis for the development of a sensitive method that can be used to obtain valuable information about the authenticity of honey.

A simple and sensitive method using pre-column high-performance liquid chromatography (HPLC) was developed for the simultaneous detection of seven sugars, which was also applied for the preliminary identification of dried shark fins. δ13C values of the dried shark fin samples also were determined by element analyser-isotope ratio mass spectrometry (EA-IRMS). The dried shark fins were decomposed by enzymolysis using a papaya protease and an amylase enzyme; then, samples were analysed by HPLC after the derivatization of the enzymatic hydrolysate, using 1-phenyl-3-methyl-5-pyrazolone (PMP) as the derivatizing agent. The results indicated that the seven sugars were well separated by this method. Of the conditions tested, application of a derivatization time of 75 minutes and of a phosphate buffer–acetonitrile (80 : 20, v/v) mobile phase system (pH = 6.8) yielded the best detection results and separation effects of the seven standard substances. Note that the areas under the peaks of two kinds of uronic acid derivative products decreased significantly (P < 0.01) with time, so it is necessary to perform the HPLC analysis within 24 h after the derivatization reaction. The developed method is suitable for the identification and analysis of artificial dried shark fins with good accuracy, reproducibility and sensitivity. In addition, δ13C values were highly significant (P < 0.01) for real and fake dried shark fins. Thus, the combination of these methods could potentially be useful for identification of dried shark fins regardless of whether they are fake or artificial.

Objective: We assessed a microfluidic method of sperm selection. Methods: Sperm was collected from 33 infertile, but otherwise healthy donors. We established a microfluidic device consisting of a 250 μm diameter microchannel of 3 cm in length, to mimic the oviduct. Human tubal fluid was pumped through the tubule at 500 μm s−1, and 5 ml sperm was added. The movement of sperm was observed for 30 min using a Live Cell Imaging System. Sperm motility was assessed by using a Computer-aided Sperm Analysis System, morphological parameters were observed by conventional light microscopy and transmission electron microscopy, and DNA integrity was measured by comet assay. We compared the fitness of sperm selected using a microfluidic device with pre-selection sperm and sperm selected by the density gradient and swim-up method. Results: Microfluidic selection improved motility parameters of all 33 samples. Sperm selected by using the microfluidic device had higher rates of average ratio of sperm fast swimming forward average sperm activity ratio, value derivation for the curvilinear, value derivation for straight-line velocities, value derivation for average path, amplitude of lateral head displacement, amplitude of cross-frequency, progression ratios of linearity, progression ratios of wobble, progression ratios of straightness, and lower rates of deformity than sperm selected using conventional methods (p < 0.05). Furthermore, the DNA integrity of sperm selection by our device was better maintained than that selected by traditional methods (p < 0.05). Conclusion: This method can not only minimize damage to the sperm structure, but also increase the effectiveness of selection.

In recent years, the safety problem of infant formula powder has provoked panic among Chinese citizens. One of the most notorious incidents was the 2008 Chinese milk scandal which harmed thousands of infants. Therefore the development of a fast, high throughput screening method towards melamine and other harmful chemicals is of paramount importance. Microsphere-based flow cytometry is a new multiplexing immunochemical method for food-testing. Through competition between the free analyte in the sample and analyte-coupled microspheres, the content of the analyte can be indirectly quantified by measuring the fluorescence emitted from labelled antibodies. This developed method of high sensitivity (limit of detection = 0.70 ng ml−1) has already been validated and successfully applied to screen for melamine in various brands of infant formula powder analysis fulfilling the legislated tolerance level in a simple, fast, high throughput and organic solvent-free manner.

An on-line solid phase extraction coupled with ultra-high performance liquid chromatography in tandem with mass spectrometry (SPE-UHPLC-MS/MS) method for the determination of fourteen hormones (four oestrogens, three androgens, four progestogens and three corticosteroids) in waste water samples has been developed. All of the parameters involved in the on-line extraction process have been optimized: type of cartridge, sample volume, loading solvent, solvent of the wash step and the pH of the sample. Moreover, the chromatographic separation and all of the parameters involved in the detection by mass spectrometry have been studied too. The developed method allows for complete analysis (extraction and identification of the analytes) in 14.5 minutes. The method is selective, with satisfactory relative standard deviations (lower than 15% in most cases) and limits of detection and quantification that ranged from 0.5 to 13.2 ng L−1 and from 1.66 to 44 ng L−1, respectively. The recoveries were acceptable for most compounds for effluent samples from different waste water treatment plants (between 50 and 90%). The proposed method has been applied to study effluent samples from three waste water treatment plants from Gran Canaria (Spain). Four steroid hormones of different families have been detected at concentrations ranging from 3.1 to 52.8 ng L−1.

Monitoring the wheat straw fermentation process using an electronic nose with pattern recognition methods by Congli Mei; Ming Yang; Dongxin Shu; Hui Jiang; Guohai Liu (6006-6011).
To monitor the wheat straw solid-state fermentation process in real time, an electronic nose (e-nose) was attempted in this study. The e-nose was designed to detect gas changes in the fermentation process and was equipped with a sensor array composed of eleven selected commercially available metal oxide semiconductor (MOS) gas sensors. Using the e-nose data, an appropriate monitoring model can be constructed to determine process states. Therefore, selecting the optimal pattern recognition method was crucial. For the simplicity of monitoring models, principal component analysis was used to extract features (i.e. principal components or latent variables) of the e-nose data as inputs of monitoring models. For comparison, three representative methods (i.e. Gaussian process, support vector machine and back propagation neural networks) were assessed. The results sufficiently demonstrated excellent promise for the e-nose technique and the Gaussian process performed better than the other two pattern recognition methods.

In this study an adsorbent, a magnetic zeolite modified with 2-(3,4-dihydroxyphenyl)-1,3-dithiane, was synthesized as an easily separable sorbent for the simultaneous removal of two toxic heavy metals, cadmium and copper, from soil and water samples. The synthesized magnetic sorbent was characterized by SEM and XRD. The magnetic properties of the sorbent were identified by the VSM method. The obtained saturation magnetization of 18.4 emu g−1 showed a facile separation of the magnetic modified zeolite after the adsorption process. The effects of the five dominant parameters of pH, temperature, time, amount of sorbent and sample solution volume on the adsorption process were investigated. The optimum conditions of 6, 25 °C, 9 min, 40 mg and 40 mL were acquired for pH, temperature, time, amount of sorbent and sample solution volume, respectively. Maximum experimentally achieved adsorption percentages of 98.2 ± 2.5 and 97.5 ± 2.8 were obtained under the optimum conditions which showed the high adsorption potential of the proposed sorbent. The experimental data were found to fit properly to the Langmuir and Freundlich models which indicated that the sorption took place on a heterogeneous material. Sorption capacities of 178.5711 and 181.8182 (mg g−1) were achieved for cadmium and copper respectively from sorption isotherms. A three-layer artificial neural network model with 8 neurons and a tan-sigmoidal function at the hidden layer and a linear transfer function (purelin) at the output layer was developed to predict the simultaneous removal of cadmium and copper. The results indicated that the proposed artificial neural network model could perfectly predict the process with a mean square error (MSE) of 0.037. The optimization procedure showed a close correlation between the experimental and predicted values.

Rapid fingerprinting of Rauwolfia species using direct analysis in real time mass spectrometry combined with principal component analysis for their discrimination by Sunil Kumar; Vikas Bajpai; Awantika Singh; S. Bindu; Mukesh Srivastava; K. B. Rameshkumar; Brijesh Kumar (6021-6026).
Medicinal plants of the genus Rauwolfia (Apocynaceae) are extensively used as folk medicines worldwide. Its antihypertensive activity is well known due to the presence of monoterpene indole alkaloids (MIAs). The therapeutic potential of the herbal medicines are affected due to variation of bioactive phytoconstituents. Therefore, a rapid and validated method was developed for fingerprinting of roots and leaves of six Rauwolfia species by direct analysis in real time mass spectrometry (DART-MS). Seventeen bioactive MIAs were tentatively identified on the basis of their exact mass measurement from the intact plant parts. Furthermore, principal component analysis (PCA) was used to analyze the DART-MS data of six Rauwolfia species to identify the chemical markers. Thirteen and twenty-three chemical markers were identified from the roots and leaves which were able to discriminate among six Rauwolfia species. This method was also cross-validated for the rapid identification, authentication and quality control of Rauwolfia species.

A versatile probe for chemoselective capture and analysis of carbonyl compounds in exhaled breath by Ralph J. Knipp; Mingxiao Li; Xiao-An Fu; Michael H. Nantz (6027-6033).
We describe an aminooxy reagent for the capture of trace aldehyde and ketone volatile organic compounds (VOCs) in exhaled breath. The reagent, 4-(2-aminooxyethyl)-morpholin-4-ium chloride (AMAH), when coated onto micropillars within a silicon microreactor, chemoselectively and covalently retains carbonyl VOCs from exhaled breath. The AMAH–carbonyl adducts are then eluted from the microreactor with methanol and directly analyzed by Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry (MS), where the aminium ion of the reagent enhances the sensitivity for high mass accuracy. We also outline a protocol for treatment of the AMAH–carbonyl adducts with poly(4-vinylpyridine) to afford the corresponding volatile carbonyl adducts that now can be analyzed by gas chromatography-mass spectrometry (GC-MS). This convenient protocol imparts flexibility for the identification and quantification of isomeric VOCs using both FT-ICR-MS and GC-MS. Representative breath analyses are given to illustrate this applicability of AMAH.

Back cover (6037-6038).