Analytical Methods (v.6, #12)

Front cover (3867-3868).

Contents list (3869-3888).

Analytical methods for clinical diagnostics by S. Garrigues; M. de la Guardia (3889-3890).

Nanomaterial-based electrochemical immunosensors as advanced diagnostic tools by Jafar Ezzati Nazhad Dolatabadi; Miguel de la Guardia (3891-3900).
A literature survey has been made of recent advances in the use of new nanomaterials for the biosensing of different biomolecules, which have opened new horizons in identifying and quantifying biomolecules, for possible early diagnosis of disease. Nanomaterials show similar dimensions to biomolecules like proteins (enzymes, antigens and antibodies) and DNA. The integration of nanomaterials with biomaterials results in novel hybrid nanobiomaterials with synergetic properties and functions. In this review, we will discuss recent progress in the field, describing the basic concepts of nanomaterial-based platforms for development and expansion of immunosensors and nanosensors. We present an overview of the various nanomaterial-based immunosensors that utilize different immune reagent recognition elements for nanomedical applications.

Vibrational spectroscopy for cancer diagnostics by O. J. Old; L. M. Fullwood; R. Scott; G. R. Lloyd; L. M. Almond; N. A. Shepherd; N. Stone; H. Barr; C. Kendall (3901-3917).
The vibrational spectroscopy techniques of Raman spectroscopy and Fourier-transform infrared spectroscopy offer a number of potential advantages as tools for clinical diagnosis. The ability of these methods to detect subtle biochemical changes relating to pathology opens the possibility of their use in tissue diagnosis. Potential applications include use as an ‘optical biopsy’ technique for in vivo tissue diagnosis or to guide therapy, as a ‘digital staining’ method to assist a histopathologist in analysing a sample, or as an entirely automated process for histopathology classification. To date, much work has been undertaken in applying these spectroscopic methods to discriminate between disease states across a wide range of pathologies and organ systems, but as yet none have entered routine clinical practice. There is a pressing clinical need for real-time, accurate tissue diagnosis, especially in malignant conditions for which rapid diagnosis and comprehensive identification and treatment of diseased tissue are of paramount importance. Cancer diagnostics remains reliant on analysis of tissue samples by histopathologists to confirm malignancy, based on morphological tissue changes and immunohistochemical staining techniques. There is increasing evidence that vibrational spectroscopy, in combination with chemometric data analysis, is a powerful and accurate technique for detecting cancerous and pre-cancerous biochemical changes both in vitro and in vivo, for a range of malignant conditions. This review examines the progress of vibrational spectroscopy towards selected clinical applications, with a particular focus on cancer diagnostics.

Cancer glycan biomarkers and their detection – past, present and future by Sergei A. Svarovsky; Lokesh Joshi (3918-3936).
Glycans are important in most biological processes, yet the exact insights into their functions remain enigmatic due to the lack of suitable tools for their analysis. Glycoproteins have recently emerged as valuable biomarkers for a wide range of diseases and some of them (e.g. AFP-L3) have already been approved by the US Food and Drug Administration for cancer diagnosis. Increasing attention is now being placed on the examination of glycan modifications because they can increase the predictive values of glycoprotein biomarkers. The main challenge to implementation of glycan biomarkers in clinical settings, however, remains the availability of suitable analytical methods for their identification and detection. The ability to rapidly detect glycan biomarkers that are indicators of pathology such as inflammation, infection and cancer with high sensitivity and specificity using glycobiosensors will without doubt offer better tools for early-stage disease diagnosis. In this critical review we first provide a brief overview of the existing technologies for glycobiomarker identification and then discuss methods for their detection focusing on the advances in microarray technologies made in the past few years. Current challenges and perspectives on the emerging and future technologies that may help springboard this important field from the academic domain to viable diagnostic tools are discussed.

Metabolite fingerprint analysis of cervical cancer using LC-QTOF/MS and multivariate data analysis by Qun Liang; Qian Yu; Haikun Wu; Yong-zhi Zhu; Ai-hua Zhang (3937-3942).
Cervical cancer (CC) is the second most common cancer in females worldwide, as yet, the metabolic alterations that are specific for the development of CC have not been fully determined, which also precludes the early diagnosis and prognosis of this pathology. In this pilot study, we determined the metabolic fingerprint of urine samples from women diagnosed with CC and women not diagnosed with CC using LC (Agilent 1290 Infinity LC System) coupled with Q-TOF/MS (Agilent, 6550 iFunnel) and independent variable analysis. Urine fingerprints allowed for the discrimination of women diagnosed with CC from the control subjects. In addition, we identified a set of metabolites with a strong discriminative power, such as 3-methylhistidine, citric acid, cytosine, indoleacetic acid, salicyluric acid, l-methionine, aminomalonic acid, glutaric acid, ursodeoxycholic acid and N-acetylornithine, which are involved in key metabolic pathways such as the citrate cycle, lysine degradation, tryptophan metabolism, cysteine and methionine metabolism, etc. Finally, we provide evidence for the implication of these compounds in metabolic routes that may be associated with the early genesis of CC, which highlights their potential use as prognostic markers for the identification of women at risk of developing CC. Urine fingerprints reveal disease-specific metabolic imbalances in women diagnosed with CC .

LOC-SERS: towards point-of-care diagnostic of methotrexate by I. J. Hidi; A. Mühlig; M. Jahn; F. Liebold; D. Cialla; K. Weber; J. Popp (3943-3947).
Therapeutic drug monitoring is of major importance in the case of medication with a narrow therapeutic range as well as when pharmacokinetic/pharmacodynamic variability is suspected. Methotrexate (MTX), an antifolate antibiotic, proved to be toxic regardless of the chosen treatment schedule. In this contribution, a new analytical method was used for the detection of MTX. A linear response was achieved in the 0.2–2 μM concentration range, with a limit of detection ≈ 0.17 μM. The lab-on-a-chip surface enhanced Raman spectroscopy (LOC-SERS) approach combines the fingerprint specificity and high sensitivity of SERS with the high sample throughput of a microfluidic platform. Additionally, it is shown that due to the chemical affinity of the MTX molecules towards Ag nanostructures, the pH value of the solving medium highly affects the obtained SERS signal. More specifically, SERS signals with well resolved bands can be obtained from deprotonated MTX molecules due to their binding to the metallic surface via the amine groups of the aromatic ring.

Investigating the use of Raman and immersion Raman spectroscopy for spectral histopathology of metastatic brain cancer and primary sites of origin by Leanne M. Fullwood; Graeme Clemens; Dave Griffiths; Katherine Ashton; Timothy P. Dawson; Robert W. Lea; Charles Davis; Franck Bonnier; Hugh J. Byrne; Matthew J. Baker (3948-3961).
It is estimated that approximately 13 000 people in the UK are diagnosed with brain cancer every year; of which 60% are metastatic. Current methods of diagnosis can be subjective, invasive and have long diagnostic windows. Raman spectroscopy provides a non-destructive, non-invasive, rapid and economical method for diagnosing diseases. The aim of this study was to investigate the use of Raman and immersion Raman spectroscopy for diagnosing metastatic brain cancer and identifying primary sites of origin using brain tissue. Through spectral examination, the Raman peaks at 721 cm−1 and 782 cm−1 were identified as being the most distinct for discriminating between the glioblastoma multiforme (GBM), metastatic and normal brain tissue spectra. A ratio score plot of these peaks calculated the classification sensitivities and specificities as 100% and 94.44% for GBM, 96.55% and 100% for metastatic brain, and 85.71% and 100% for normal brain tissue. Principal Component-Linear Discriminant Analysis (PC-LDA) also showed discrimination between normal, GBM and metastatic brain tissue spectra. We also present, for the first time, the use of Raman spectroscopy to investigate primary site of origin for metastatic brain cancer and any biochemical differences between different primary and metastatic cancer using linked samples. This study revealed interesting spectral differences in the amide regions showing changes in the biochemistry of the metastatic brain cancer from the primary cancer.

Locating microcalcifications in breast histopathology sections using micro CT and XRF mapping by Robert Scott; Catherine Kendall; Nicholas Stone; Keith Rogers (3962-3966).
Spectroscopic measurement of microcalcification chemistry holds great promise as a rapid, quantitative, and non-invasive aid to diagnosis of early stage breast cancer. Previous work has shown that carbonate substitution in hydroxyapatite is highly correlated to breast cancer grade. A deeper understanding of the chemistry–pathology relationships is important in the development of spectroscopic aids to diagnosis. However, investigation of calcification chemistry is hampered by the difficulty of quickly and systematically locating microcalcifications within tissue specimens. We have demonstrated two simple methods based on micro-CT and XRF mapping which can achieve this in sections cut from wax embedded breast tissue from diagnostic archives.

Carbohydrate deficient transferrin (CDT) is a biochemical marker for congenital disorders of glycosylation (CDG), chronic alcohol consumption, and forensic medicine diagnosis. However it is necessary to take into account that CDT is not a single molecular entity but refers to a group of transferrin (Tf) sialoforms (asialo-, monosialo-, disialo- and occasionally trisialo-Tf). A number of methods have been developed for CDT measurement based on different analytical techniques and principles without harmonization or calibration to a reference method or a certified reference material, hampering understanding of the diagnostic value of CDT and its routine use. Thus, it is unquestionable that there is a need for a reference material which permits the accurate and precise determination of each individual Tf sialoform which could serve as a universal calibrator for routine immunologic methods used in clinical laboratories. In this work, we describe highly sensitive ICP-MS isotope dilution analysis (IDA) methods for the separation and quantification of the different Tf sialoforms in human serum. The methodology was applied to measure the concentration of each sialoform of Tf and the total concentration of Tf in the NIST Standard Reference Material (SRM) 909c human serum. Additionally, two clinical laboratory control serums utilized for routine analysis of CDT were also analyzed. The separation of the sialoforms was achieved by anion exchange chromatography. The two IDA techniques applied for the quantification of the Tf sialoforms were: (a) post-column IDA and (b) species-specific IDA with the total concentration of Tf calculated by these two being the sum of the individual sialoforms. A third IDA technique, exact matching IDA was applied to determine the total concentration of Tf in SRM 909c. All the Tf measurements were validated with the ERM-DA470-IFF human serum (IRMM, Geel, Belgium) certified for total Tf. Finally, the identification of each Tf sialoform previously separated in the serum (SRM 909c) was carried out by LC/MS/MS.

High density lipoprotein cholesterol (HDL-C) is a modifiable risk factor in cardiovascular disease and devices suitable for its determination at the point of care are critical to the future management of hypercholesterolaemia. An electrochemical biosensor for measuring HDL-C was developed. The biosensor was based on a homogeneous assay methodology for selective determination of HDL-C in combination with a printed electrochemical sensor for measuring the reduction of hydrogen peroxide at a silver paste electrode. The polyoxyethylene alkylene tribenzylphenyl ether surfactant (Emulgen B-66) was found to be capable of both the selective dissolution of HDL particles, as well as the enhanced electrocatalytic reduction of hydrogen peroxide. The resulting biosensor was shown to have a linear response to HDL-C from 0.5 to 4 mM (r2 = 0.998) with an average r.s.d. of 7%. The biosensor was also used to analyse HDL-C in thirteen serum samples and had good agreement with a commercial spectrophotometric precipitation-based assay (r = 0.72; p < 0.058).

Determination of biochemical parameters in human serum by near-infrared spectroscopy by J. L. García-García; D. Pérez-Guaita; J. Ventura-Gayete; S. Garrigues; M. de la Guardia (3982-3989).
NIR offers multiple advantages for serum analysis, permitting a fast and direct determination of several parameters simultaneously, with low sample handling and without the need for reagents during the measurement step. The aim of this paper was to provide an evaluation of this technique in a real world scale, for the simultaneous determination of several parameters and based on a considerable number of samples. Direct near infrared (NIR) absorbance measurements were used to determine the concentration of clinical parameters in human serum that are required in routine biochemical tests. Total protein, albumin, total cholesterol, high-density lipoprotein (HDL cholesterol), low-density lipoprotein (LDL cholesterol), and very low-density lipoprotein (VLDL cholesterol), triglycerides, urea and glucose were determined in 447 serum samples obtained randomly from the clinical laboratory of the University Hospital Doctor Peset in Valencia (Spain). NIR spectra from 12 500 to 4000 cm−1 obtained with a 1 mm optical path length were evaluated by using partial least squares regression models (PLS) built from the spectra of samples with known concentrations provided by the hospital. Root mean square error cross-validation (RMSECV) was used for selecting a number of factors, spectral regions and spectral preprocessing considered to build the models, that were evaluated from their prediction capability using the relative root mean square error of prediction (RRMSEP) of a series of around 30 independent samples, not used for calibration. For some analytes such as total protein, albumin, total cholesterol and triglycerides, errors obtained were 2.3, 4.4, 5.1, and 6.2% respectively, evidencing that the proposed methodology could compete with the enzymatic reference methodologies. However in the case of urea, glucose, HDL and LDL, average errors obtained were 16.0, 16.2, 18.0 and 11.0% respectively, and therefore the NIR methodology proposed is limited as a screening tool. With the use of a considerable number of samples for calibration, this study confirms that the proposed green and cost-effective methodology is ready for scaling up from the bench to the real world.

Determination of methylarginines in infant plasma by CE-LIF by Thomas H. Linz; Susan M. Lunte (3990-3994).
Methylarginines (MAs) are a class of nitric oxide synthase inhibitors that have been implicated in respiratory complications of critically ill infants. This paper describes the development of an analytical method to measure these compounds in the plasma of newborns using capillary electrophoresis (CE). The CE separation method was optimized to enable complete baseline resolution of the four MA analogues of interest. Sample preparation concerns for infant-derived samples were addressed by validating a heat-assisted extraction method for the analysis of low volume (≤100 μL) samples from a plasma matrix. It was determined that the sample matrix (plasma versus serum) did not affect the measured MA concentrations, while extracting smaller volumes of plasma that underwent heat-induced gelation afforded higher MA recoveries than larger volume samples. These methods were then applied to blood samples collected from infants housed in the neonatal intensive care unit. It was discovered that these newborns had significantly elevated concentrations of MAs at younger ages (<6 months) while amounts were similar between infants 6 months old and adults. The data are preliminary, but demonstrate an interesting age dependence on the concentrations of these nitric oxide inhibitors, which has not been previously reported.

Protein/peptide purification by three-well OFFGEL electrophoresis with immobilized ultra narrow pH gradient gels by Elena Tobolkina; Fernando Cortés-Salazar; Liang Qiao; Hubert H. Girault (3995-4002).
Purification and desalting of protein and peptide samples by three-well OFFGEL electrophoresis with immobilized ultra narrow pH gradient gels is proposed as a fast preparative strategy for proteomics. The gist of this strategy is to separate proteins and peptides according to their isoelectric points and to isolate those of a given pI value equal to the mean pH value of the gel. The present approach has been demonstrated both on protein mixtures and a digested Escherichia coli protein extract. UV-Vis spectroscopy, MALDI-MS, SDS-PAGE and LC-MS/MS were employed for the quantitative and qualitative characterization of the separation results. The electrophoretic methodology has been simulated by finite element methods.

Determination of boron in silicon without use of additional complexing agents by Matthias Balski; Franziska Emmerling; Heinrich Kipphardt; Ulrich Panne (4003-4008).
Methods for the determination of boron in various matrices described in the literature usually employ complexing agents like mannitol to retain the volatile boron species during matrix evaporation steps. However, also relatively high boron recoveries from silicon containing samples have been reported when no complexing agents were added to the digestion acids. The mechanism behind this matrix-dependant recovery has been investigated by studying the boron recovery in the analysis of solar grade silicon. It was found that the NH4+ ion formed by the reduction of nitric acid during sample digestion is responsible for the higher recovery of boron, which leads to a possible analysis method without the use of complexing agents if the sample preparation procedure is carefully optimized.

Characterisation and classification of binders used in art materials at the class and the subclass level by R. Checa-Moreno; E. Manzano; L. F. Capitán-Vallvey (4009-4021).
SIMCA pattern recognition is used with amino acid chromatographic profiles in a large homemade collection of natural protein binders obtained following old recipes traditionally used by painters and considered here as the standard of classification. An initial cluster analysis of the full dataset made it possible to distinguish three main classes of protein binders: albumin, casein and collagen-like substances. An additional iterative study of each class revealed a new subclass, i.e., glair, yolk and whole egg for the albumin class; goat, sheep and cow for the casein class; and mammals and fish for the collagen class. Optimized SIMCA models for each class and subclass were obtained with good results in terms of sensitivity (90–100%), specificity (73–100%) and interclass distance (>1.4), providing identification of the protein binder present in a set of samples of different origins such as natural products, commercial binders and works of art considered cultural heritage.

Photochemical derivatization of amitriptyline using a green chemistry approach: fluorimetric determination and photochemical reaction mechanism by Ana C. P. Osorio; Alessandra L. M. C. da Cunha; Sarzamin Khan; Caroline J. Franco; Annibal D. Pereira-Netto; Ricardo Q. Aucélio (4022-4028).
Intense fluorescence from amitriptyline (AMT) was achieved after photochemical derivatization in an acidic aqueous medium (60 min of UV irradiation of solutions containing 0.027 mol L−1 HCl). After the reaction, fluorescence was significantly enhanced enabling the indirect fluorimetric determination of AMT in drugs and in a pharmacopoeia reference material. The limit of quantification (LOQ) was 1.3 × 10−7 mol L−1 and measurement uncertainty was about 6% at the concentrations close to the LOQ and less than 4% at higher concentrations. The mechanism of the photoreaction was proposed based on the identification of the products and on its kinetics. Studies aiming at the determination of AMT in human serum indicate the potential of the approach as a cheap and simple screening procedure for tricyclic antidepressants.

A bi-functionalized molecularly imprinted silica (MIS) was prepared for solid phase extraction of quercetin. To ensure the specificity of molecular recognition, aluminum ions were inserted in the silica matrix to form Lewis and Brønsted acid sites and allow the interaction with quercetin, as well as with 3-aminopropyltriethoxysilane as a functional monomer. A sol–gel process was used to prepare MIS–Al with tetraethoxysilane as the crosslinker reagent. The efficiency of imprinted silica was evaluated by comparing the amount of quercetin adsorbed by MIS–Al with those by non-imprinted silica (NIS–Al) and control polymers without Al. The adsorption capacity and extraction efficiency were studied using different solvents and a mixture of ethanol–water (60 : 40, v/v) was found to be most effective for the binding of quercetin with MIS–Al while pure ethanol was most effective for extraction. The selectivity was evaluated by HPLC using a mixture of quercetin and rutin, a molecule which was considered to have an analogous structure. Characterization of the imprinted silica and adsorption capacity tests suggested that the MIS–Al had a higher adsorption capacity and reproducibility than MIS without Al (248.5 ± 3.5 against 159.7 ± 35.0), proving that the presence of aluminum ions improved the selectivity and efficiency for quercetin extraction. Herbal medicine samples of Ginkgo biloba L. capsules (40 and 80 mg) were analysed by passing the extract through an SPE–MIS–Al cartridge and the fractions collected were analysed by HPLC-PDA. The MIS–Al was shown to be selective and resulted in cleaner chromatograms with better resolution of the quercetin peak, proving this to be an effective clean-up step before chromatographic analysis. By the proposed method, it was found that 40 and 80 mg capsules contained 0.25 ± 0.01 mg and 0.24 ± 0.01 mg quercetin per capsule of Ginkgo biloba L., corresponding to 2.6% and 1.3% (w/w), respectively. These results are in agreement with scientific studies in the literature that reports a range of 0.5 to 4.7% mg of quercetin per capsule.

Quantitative analysis of bromate in non-alcoholic beer using ultra performance liquid chromatography-electrospray ionization mass spectrometry by Mohammad Rizwan Khan; Zeid Abdullah Alothman; Nasser Judaya Alqahtani; Ibrahim Hotan Alsohaimi; Mu Naushad (4038-4045).
Bromate (BrO3) is a possible human carcinogen which is formed as a bromide containing source water ozone disinfection by-product. Thus, BrO3 is subject to assess its threat to humans. In the present study, an ultra performance liquid chromatography-electrospray ionization mass spectrometry method has been developed for the quantitative analysis of BrO3 in non-alcoholic beer. The chromatographic separation of both transitions 81BrO3 and 79BrO3 (isotope contributions of 79Br and 81Br) was carried out and achieved in <1 min, with superior peak symmetry. The good quality parameters were obtained such as linearity (R2 > 0.9999), precision (repeatability and reproducibility in terms of relative standard deviation) <1% at 1.0 μg mL−1 standard and <3% in the analysis of sample 1 (classic malt) and sensitivity with a low limit of detection (LOD, 81BrO3, 0.03 ng mL−1 and 79BrO3, 0.03 ng mL−1) and limit of quantification (LOQ, 81BrO3, 0.1 ng mL−1 and 79BrO3, 0.1 ng mL−1) in the standard. Nevertheless, the LOD and LOQ in sample 1 (classic malt) were obtained only for transition 81BrO3, 0.05 ng mL−1 and 0.16 ng mL−1, respectively. For transition 79BrO3 the obtained peak was below the limit of detection. A total of 39 non-alcoholic beer samples of different flavors were analyzed and the 81BrO3 level in classic samples was found between 1.20 μg mL−1 and 3.73 μg mL−1, however, in other flavored samples the concentration levels reached up to 14.04 μg mL−1 with excellent recovery rates (96–99%). In most of the analyzed real samples, only transition 81BrO3 was found nevertheless, transition 79BrO3 was either <LOD or completely diminished from the obtained sample chromatogram. This is one of the main findings from the present study and the mechanism of this cause is unknown. The obtained BrO3 levels were much higher than the limits in drinking water (10–25 ng mL−1) recommended by several regulatory agencies.

Protein fingerprinting in the choice of cellulase cocktails for the conversion of lignocellulosic biomass by Chiara Cattaneo; Stefano Spertino; Lara Boatti; Sara Icardi; Maria Cavaletto (4046-4055).
Based on two-dimensional electrophoresis and LC-MS/MS analysis, “protein fingerprinting” is a powerful approach for protein identification. Here we describe the application of this approach for the characterisation of cellulase cocktails. We report the protein map of a natural secretome, derived from P. decumbens, a fungal strain grown on lignocellulosic biomass; for comparison, protein maps for two commercial mixtures, Celluclast 1.5L and Novozymes SP188, are presented. A secretome protein map derived from P. decumbens can potentially serve as a guide for the design of commercial mixtures; it can be used to develop a standard for quality control to monitor available commercial enzymatic mixtures.

A simple method addressing the problem of minimizing the prediction relative error is proposed for multivariate calibration. The method is based on the use of back-propagation artificial neural network (BP-ANN). The regression objective of the simple method is to minimize the prediction relative error by changing the output values of BP-ANN. With both theoretical support and analysis of near infrared spectroscopic data and ultraviolet spectroscopic data, it is demonstrated that the simple method produced lower prediction relative error than partial least squares (PLS), principal component regression (PCR), and BP-ANN methods for the system with a wide content range. In addition, when we consider the value of the root mean square error of prediction (RMSEP), four methods were found to have a similar prediction performance. The simple method can predict low content more accurately for the system with a wide content range.

In this study, hemoglobin (Hb) was entrapped into the exfoliated Ni–Al–CO3 layered double hydroxides (LDH). UV-vis spectra analysis displayed that no significant denaturation occurred to the protein. Electrochemical results showed that exfoliation of LDH enhanced the direct electron transfer between protein molecules and electrode, and the entrapped protein showed high bioactivity in a wide range of pH values. A pair of well-defined redox peaks was observed at −0.39 and −0.33 V on the glassy carbon electrode (GCE) modified with the Hb–LDH composite. The electrode reactions showed a surface-controlled process with single electron transfer at the scan rate from 100 to 400 mV s−1. The sensor constructed displayed an excellent response to the reduction of hydrogen peroxide (H2O2) with a wide linear range, low detection limit and good stability. The modified electrode can also be used for the reduction of oxygen.

Partition of proteins with extraction in aqueous two-phase system by hydroxyl ammonium-based ionic liquid by Jing Chen; Yuzhi Wang; Qun Zeng; Xueqin Ding; Yanhua Huang (4067-4076).
A series of hydroxyl ammonium ionic liquids (ILs) have been designed and synthesized. An ionic liquid aqueous two-phase system based on N,N-dimethylethanolamine propionate ([DMEA][Pr]) ionic liquid was studied for the extraction of proteins. Based on the single-factor experiment, an initial serial investigative test was used to identify the optimal conditions. Five factors and four levels of orthogonal experiments were used to verify the optimum extraction conditions. The results show that under the optimum conditions, the extraction efficiency could reach up to 99.50%. The RSD of extraction efficiencies in precision experiment, repeatability experiment and stability experiment were 0.3% (n = 5), 1.1% (n = 5) and 1.5% (n = 5), respectively. The conformation of the proteins was not affected after extraction into the IL-rich phase in terms of the determination by UV-vis and FT-IR spectra. According to the determination of conductivity, dynamic light scattering, and transmission electron microscope images, the microstructure of the IL-rich phase and the possible mechanism for the extraction are investigated. Hydrogen bonding interaction, salt out effect and the aggregation phenomenon played important roles in the extraction. The circular dichroism spectral experiment analysis indicated that the secondary structures of the protein were unchanged after extract. Based on these findings, it is suggested that the method of hydroxyl ammonium-based ILs-ATPs have the potential to offer new possibility in the extraction of bio-analytes.

Surface-enhanced Raman scattering on a zigzag microfluidic chip: towards high-sensitivity detection of As(iii) ions by Nan Qi; Bowei Li; Huiyan You; Wei Zhang; Longwen Fu; Yunqing Wang; Lingxin Chen (4077-4082).
In this study, a microfluidic platform was combined with surface-enhanced Raman scattering (SERS) to implement the rapid quantitative detection of As(iii) ions in a continuous flow. Silver nanoparticles (AgNPs) were used as the SERS enhancement substrate, and glutathione (GSH) with 4-mercaptopyridine (4-MPY) was conjugated on the surface of the AgNPs. When As(iii) ions encountered GSH/4-MPY functionalized AgNPs, the original monodispersed probes would aggregate because As(iii) ions had a strong affinity to the GSH. As a result, Raman signals of 4-MPY adsorbed on the surface of the AgNPs were improved and the As(iii) ions could be detected. Due to the advantages of microfluidics technology combined with SERS detection, the highly sensitive and reproducible analysis of As(iii) ions was realized in several minutes. The proposed method allowed the quantitative analysis of As(iii) ions with a linear range (3 to 200 ppb), and the limit of detection (LOD) of the As(iii) ions was determined to be 0.67 ppb. The real water sample was also analyzed to confirm the practicability of the method and the consumption of several microliters of the sample was found to be environmentally friendly. This method also showed great potential in applying SERS combined with a lab-on-a-chip technique in the area of environmental monitoring with a high sensitivity and in an environmentally friendly way.

For the first time, a gas chromatography tandem mass spectrometry method in the multiple reaction monitoring mode was developed and validated for the separation and detection of 21 phthalate leachables in meter dose inhalers (MDI). The optimized method was reliable with high sensitivity and selectivity. Calibration curves were linear with correlation coefficients (R2) > 0.990, and limit of detection (LOD) values in the range of 0.1–4.2 ng mL−1 except for di(2-methoxyethyl) phthalate, di(2-ethoxyethyl) phthalate and di(2-n-butoxyethyl) phthalate, for which the LODs were around 10 ng mL−1. Recoveries ranged between 86.6 and 108.3%, and the relative standard devision values of precision were within 7.72%. For the five MDI batches analyzed, 5 out of 21 phthalates were detected in each sample, including dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, dibutyl phthalate and di(2-ehtylhexyl) phthalate, with total phthalate amounts of less than 260 ng per canister.

A novel and label-free method for micrococcal nuclease (MNase) detection has been presented based on single-stranded DNA (ssDNA)-scaffolded fluorescent silver nanoclusters (AgNCs). The ssDNA was introduced as the substrate for MNase and also as the scaffold for the synthesis of the AgNCs. In the absence of MNase, the ssDNA was not digested. As a result, the fluorescent AgNCs were formed and exhibited strong fluorescence. In the presence of MNase, the DNA was digested, which prohibited the formation of the AgNCs due to the lack of the DNA scaffold, resulting in weak fluorescence. The fluorescence intensity exhibits a linear correlation to MNase concentration in the range of 0 U mL−1 to 2 × 10−4 U mL−1 with a detection limit of 8 × 10−6 U mL−1. Given its simplicity, easy operation, sensitivity and cost-effectiveness, this method can be extended to other nuclease assays.

A luminescent nanocrystal metal–organic framework (NMOF1) of [Cd(atc)(H2O)2]nhas been synthesized by the reaction of Cd(ii) ions with the sodium salt of H2atc (2-aminoterephthalic acid) in aqueous solution. The obtained fluorescent porous material has been characterized by X-ray diffraction, transmission electron microscopy, confocal microscopy, UV-visible spectroscopy, photoluminescence spectroscopy and surface area analysis. The synthesized NMOF1 exhibits reasonably good fluorescence characteristics (excitation wavelength = 340 nm, emission wavelength = 436 nm). The potential of the above Cd(ii) based nanocrystal metal–organic framework (NMOF1) for the sensing of the nitroaromatic-containing organophosphate pesticides (nitro OPs) parathion, methyl parathion, paraoxon and fenitrothion is demonstrated. It has been possible to detect the above four OPs separately in the concentration range of 1–500 ppb. The detection limit of the proposed method for all the said OPs is 1 ppb. Interestingly, their mixture also shows the above characteristic data. The proposed method for the sensing of nitro OPs is also selective towards other OPs such as malathion, dichlorvos and monocrotophos.

SiO2 nanoparticles and diphenylcarbazide doped polymethylmethacrylate electrospun fibrous film for Cd2+ colorimetric detection by Tianyu Yao; Qin Tu; Xiang Han; Longlong Zhang; Dong-En Wang; Manlin Li; Sheng Chen; Jinyi Wang (4102-4106).
Based on the formation of a red [Cd-diphenylcarbazide] complex, a simple and visible method for Cd2+ colorimetric detection was developed by doping SiO2 nanoparticles and diphenylcarbazide in polymethylmethacrylate electrospun fibers. Benefiting from the large surface area due to the addition of SiO2 nanoparticles, the color of the fibrous film changed clearly even in the presence of extremely low concentrations of Cd2+. The detection limit was found to be 1 × 10−8 M. Investigation of the selectivity of the fibrous film on the detection of various metal ions showed that only Cd2+ could induce a yellow-to-red color change. The results demonstrated that SiO2 and diphenylcarbazide doped polymethylmethacrylate fibrous films could act as highly selective strips for the detection of Cd2+ with little interference from other metal ions.

Three amino alcohols, propranolol, clenbuterol and cycloclenbuterol, were enantioseparated on an achiral column by reversed-phase high performance liquid chromatography (RP-HPLC). A chiral chromatographic system was established based on C18-bonded silica gel as the support and a self-prepared di-n-butyl-l-tartrate–boric acid complex as the chiral mobile phase additive. In order to obtain better enantioseparations, the influences of di-n-butyl-l-tartrate and boric acid concentrations, the type, concentration and pH of the buffer, as well as methanol content were extensively investigated. It was found that the mobile phase composition played an important role in improving the enantioseparations and all of the enantiomers could be baseline resolved under the optimized experimental conditions. Three pairs of enantiomers, which could not be separated with only di-n-butyl-l-tartrate, obtained good chiral separations using the complex chiral selector. The primary driving forces responsible for the chiral recognition were assumed to be the ion-pair interaction between the enantiomers and the chiral selector; and the interactions between the ion-pairs above and the stationary phase also played important roles for the chiral separations in RP-HPLC.

Vortex-assisted emulsification microextraction followed by in-syringe ultrasound-assisted back-microextraction to determine haloacetic acids in waters by Priscilla Rocío-Bautista; Verónica Pino; Bárbara Delgado; Ana M. Afonso; Juan H. Ayala (4115-4123).
We have evaluated a vortex-assisted emulsification microextraction (VAEME) procedure followed by in-syringe ultrasound-assisted back-microextraction for the determination of nine haloacetic acids in waters of different nature, using high-performance liquid chromatography with diode array detection. The optimized method requires 600 μL of isopropyl ether as an extractant solvent and 5 mL of the water sample containing: Na2SO4 (45%, w/v) and a low pH value (<0.5). After emulsification assisted by vortex for 5 min, the droplet is separated from the water sample after centrifugation (5 min, 3500 rpm) using a syringe. This droplet is then back-microextracted in the syringe by mixing it with a low volume (50 μL) of an aqueous solution of (NH4)2SO4 (0.2 M), to ensure compatibility with the HPLC mobile phase. After 5 min of sonication, the aqueous solution containing HAAs is directly injected into the chromatograph. The method is characterized by (a) average relative recoveries of 77.7–89.0%, depending on the spiked level, (b) average enrichment factors of ∼10 for the VAEME and of ∼21 for the overall method, (c) precisions of the overall method (expressed as relative standard deviations) between 5 and 23%, and (d) average extraction efficiencies of ∼88% for the VAEME method.

This paper describes a fast and low-cost method for determination of melamine, an emerging environmental contaminant, in soil and sediment using ultrasonic-assisted extraction coupled with high performance liquid chromatography. The parameters used for sample pretreatment and chromatographic analysis were tested and optimized. The samples were extracted by 5% (v/v) ammonia in methanol with ultrasonic assistance for 5 min, followed by centrifugation and nitrogen blowing, without a cleaning procedure such as solid phase extraction. Chromatographic separation was achieved by a cyano column using acetonitrile/water (5/95, v/v) as the mobile phase containing no ion-pair reagent, and the quantification was carried out by an external standard method. An excellent linearity for melamine was obtained over the concentration range of 0.05–5 mg kg−1 with a correlation coefficient of 0.9999. The limit of detection (S/N = 3) and the limit of quantification (S/N = 10) for melamine were 0.01 mg kg−1 and 0.04 mg kg−1, respectively. The recovery for melamine ranged from 95.0% to 105.9%, with relative standard deviations (RSDs) (n = 3) of 1.6–5.3%. The proposed method was shown to be simple, rapid, cheap, sensitive and accurate, and was applied to the analysis of melamine in soil and sediment samples with satisfactory results.

Wih a view to improve the surface-enhanced Raman scattering (SERS) properties, including the sensitivity and uniformity, a simple and practical pretreatment method via optimizing the adsorption process of probe molecules and the morphology of SERS-active nanostructures was studied. Excellent SERS performances were obtained using this method such as high sensitivity (5 × 10−14 M rhodamine 6G, 1 × 10−8 M melamine), remarkable uniformity (∼9% relative standard deviation (RSD)) and reproducibility (∼10% RSD). Uniform and repeatable silver nanoparticle arrays were fabricated by an optimized filter-based method, which guaranteed the reliability of SERS detection. The SERS intensity was greatly improved by adjusting the adsorption capacity of the noble metal nanostructures, which has a linear relationship with the pH value of silver solution below 10. An accurate and reliable enhancement factor (EF) of 3.28 × 108 was calculated through the quantification of the adsorbed probe molecules. It is worth noting that the lifetime of the substrates prepared by this method could be prolonged to two months, which was definitely longer than other ordinary silver SERS substrates. The pretreatment method not only provided perfect SERS properties, but also made SERS a practical analysis tool because of its good homogeneity and ultralow detection limit.

An accurate, simple and sensitive method for simultaneous determination of total homocysteine (Hcy) and total cysteine (Cys) in human plasma has been developed and validated. Hcy and Cys play a major role in metabolism and cellular homeostasis. An elevated level of plasma Hcy is considered an important risk factor or marker for several diseases, in particular cardiovascular disease, while Cys is involved in a variety of important cellular functions such as protein synthesis, detoxification and metabolism. The proposed analytical procedure consists of reduction of title thiol dimers and unsymmetrical disulfides with tris(2-carboxyethyl)phosphine, and derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) followed by pH-mediated stacking capillary electrophoresis separation and ultraviolet-absorbance detection of Hcy-CMQT and Cys-CMQT derivatives at 355 nm. Effective baseline electrophoretic separation was achieved using a standard fused-silica capillary (effective length 91.5 cm, 75 μm id) and 0.1 mol L−1, lithium acetate buffer adjusted to pH 4.75. The limit of quantification (signal to noise ratio of 9) for derivatized Hcy and Cys in plasma was 2 μmol L−1. The calibration curves obtained for Hcy and Cys in plasma showed linearity in the range 2–20 μmol L−1, with R2 = 0.9986 and 20–300 μmol L−1, with R2 = 0.9998, respectively. The relative standard deviation of the points of the calibration curve was lower than 7%. The method can be used for a routine monitoring of total Hcy and total Cys levels in plasma of human subjects.

Cold water forced swimming stress induced metabolic alterations in rats by Chaoni Xiao; Pu Jia; Man Wu; Yajun Zhang; Shixiang Wang; Xinfeng Zhao; Xiaohui Zheng (4144-4151).
Forced swimming in cold water causes physiological and psychological stress that leads to altered biochemical homeostasis and neurobehavioral responses. In this study, plasma and urinary metabolites in Sprague-Dawley (SD) rats subjected to cold water forced swimming stress (CWFSS) were characterized using NMR in conjunction with multivariate data analysis. Metabolic analysis demonstrated that decreased levels of lipoprotein and increased levels of O-acetyl glycoprotein, glucose, lactate, pyruvate, creatine, citrate, succinate and amino acids including phenylalanine, tyrosine, glutamate, valine, isoleucine, alanine and lysine in plasma were observed in response to CWFSS. Moreover, the biochemical response to CWFSS as determined from NMR analysis of urine was characterized by an elevation of gut microbiota-related metabolites including phenylacetylglycine, 4-cresol glucuronide and indoxyl sulfate. These results suggest that CWFSS-induced metabolic perturbations were involved in the disruption of amino acid and energy metabolism as well as the alteration of gut microbiota function. This study will enhance the current knowledge of stress-induced diseases and aid in the early diagnosis of metabolic disorders in humans.

A novel sandwich electrochemiluminescence aptasensor based on molybdenum disulfide nanosheet–graphene composites and Au nanoparticles for signal amplification by Yan-Ming Liu; Min Zhou; Ying-Ying Liu; Ke-Jing Huang; Jun-Tao Cao; Jing-Jing Zhang; Gui-Fang Shi; Yong-Hong Chen (4152-4157).
In this work, we constructed a novel sandwich electrochemiluminescence (ECL) aptasensor for sensitive detection of thrombin based on molybdenum disulfide nanosheet–graphene (MoS2–GR) composites and Au nanoparticles. In the protocol, MoS2–GR composites were firstly synthesized and assembled on the glassy carbon electrode (GCE) to promote electron transfer. Subsequently, gold nanoparticles (AuNPs) were covered on the electrode to improve the immobilized amount of aptamer1 (Apt1) that could further amplify the ECL signal as well. Afterwards, Apt1 was conjugated to the electrode via Au–S bonds. Finally, the target thrombin and the quantum dot labeled aptamer2 (QD-Apt2) as signal probes were successively attached to the GCE to fabricate a sandwich ECL aptasensor. With the excellent features of the MoS2–GR composites, AuNPs, and sandwich structures, multiple signal amplification for the ECL aptasensor has been achieved. The ECL intensity depended linearly on the logarithm of the thrombin concentration in the range from 0.02 to 5.0 pM with a detection limit of 1.3 fM (S/N = 3). Furthermore, the aptasensor was successfully applied to the determination of thrombin in human plasma with the recoveries of 90.0–102.4% and the RSDs of 2.1–3.7%. The protocol could serve as a new tool for protein detection in biochemical analysis.

A novel method that combines high performance liquid chromatography (HPLC) fingerprint with blending technology was developed and validated for ensuring quality stability of Zhou's prescription extract. For fingerprint analysis, 32 peaks were selected as the common peaks by comparing the chromatograms of 15 batches of Zhou's prescription extracts. Meanwhile, these common peaks were identified by using electronic spray ion mass spectrometry. The batch, which contained herbs that were obtained from geo-authentic habitats during their best harvest times, was selected as the standard extract (SE). The quality difference between SE and 14 batches of Zhou's prescription extracts was evaluated by using the fingerprint similarity, relative deviation of content (RDC), hierarchical clustering analysis (HCA) and principal component analysis (PCA), and narrowed by applying blending technology that could lead to homogenization of high quality of the 14 batches of Zhou's prescription extracts by using nonlinear programming. According to the mode of finding difference and narrowing difference, the previous two kinds of quality control method was combined smartly to ensure the quality stability of Zhou's prescription. Finally, this method was verified and the results indicated that fingerprint similarities of 10 different blending schemes increased to the range of 0.9208 to 0.9797 from 0.7338 to 0.8925. The average RDCs of 28 index components decreased to the range of 0.1549 to 0.2790 from 0.4768 to 0.6083. The diagrams of HCA and PCA show that the 10 blending schemes were grouped with the standard Zhou's prescription extract. These results demonstrated that this new method is an efficient and reliable approach for ensuring quality stability of Zhou's prescription extract.

UPLC-TOF/MS based urinary metabonomic studies reveal mild prevention effects of MDG-1 on metabolic disorders in diet-induced obese mice by Yuan Li; Yun-Yun Zhu; Lin-Lin Shi; Lan Shen; Hai Wei; Yuan Wang; Ke-Feng Ruan; Yi Feng (4171-4180).
Metabolic disorders such as hyperglycemia, dislipidemia, and insulin resistance often occur in obese populations long before the manifestation of type 2 diabetes mellitus (T2DM), cardiovascular disease, and cancer. The prevention of metabolic disorders in obese individuals might decrease the morbidity of diabetes and other metabolic diseases. Ophiopogon japonicus is a traditional Chinese medicine used for thousands of years to treat patients with diabetes. We showed in previous work that MDG-1, a polysaccharide extracted from Ophiopogon japonicus, could treat T2DM. To investigate whether MDG-1 can prevent metabolic disorders, metabonomic methods, together with multivariate analysis, were used to evaluate the role of MDG-1 in the prevention of metabolic disorders in a high-fat, diet-induced obesity (DIO) model. Thirty-six male C57BL/6 mice (8 weeks old) were randomly divided into a control group (normal chow), a model group (high-fat chow), and an MDG-1 group (high-fat chow dosed with 300 mg kg−1 MDG-1). After 16 weeks of treatment, urinary metabonomic studies were performed using ultra-performance liquid chromatography-time of flight mass spectrometry in combination with multivariate statistical analysis. Indices of body weight, food intake, fasting and fed blood glucose, oral glucose tolerance test (OGTT), and OGTT plasma insulin were collected. MDG-1 treatment was shown to exert mild ameliorative effects on bodyweight gain, fed blood glucose levels, OGTT, and the insulin resistance of DIO mice. In addition, 21 potential biomarkers of glucose, fatty acid, phospholipid and amino acid metabolism, the tricarboxylic acid cycle and purine metabolism were identified. Based on these compounds, it is suggested that MDG-1 could reduce glucose, the glucose-related products lactic acid and N-acetyl-d-glucosamine, and lipids, thus normalizing tricarboxylic acid cycle activity while decreasing purine, hence alleviating oxidative stress in DIO mice. Although there was no obvious alteration in visible biomedical indices, invisible metabolic disturbances did occur with MDG-1 supplementation, which may lead to the possible elucidation of the action of MDG-1 against metabolic disorders.

Analysis of fluoxetine and norfluoxetine in human plasma by HPLC-UV using a high purity C18 silica-based SPE sorbent by Carlos Eduardo Domingues Nazario; Paulo Clairmont Feitosa de Lima Gomes; Fernando Mauro Lancas (4181-4187).
This paper reports on the development and validation of a simple and sensitive method that uses solid phase extraction (SPE) and liquid chromatography with ultraviolet detection to analyze fluoxetine (FLX) and norfluoxetine (NFLX) in human plasma samples. A lab-made C18 SPE phase was synthesized by using a sol–gel process employing a low-cost silica precursor. This sorbent was fully characterized by nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM) to check the particles' shape, size and C18 functionalization. The lab-made C18 silica was used in the sample preparation step of human plasma by the SPE-HPLC-UV method. The method was validated in the 15 to 500 ng mL−1 range for both FLX and NFLX using a matrix matched curve. Detection limits of 4.3 and 4.2 ng mL−1 were obtained for FLX and NFLX, respectively. The repeatability and intermediary precision achieved varied from 7.6 to 15.0% and the accuracy ranged from −14.9 to 9.1%. The synthesized C18 sorbent was compared to commercial C18 sorbents. The average recoveries were similar (85–105%), however the lab-made C18 silica showed fewer interfering peaks in the chromatogram. After development and validation, the method using the lab-made C18 SPE was applied to plasma samples of patients under FLX treatment (n = 6). The concentrations of FLX and NFLX found in the samples varied from 46.8–215.5 and 48.0–189.9 ng mL−1, respectively.

(−)-Quinine was used as a chiral selector by adopting three approaches in TLC for direct enantioresolution of dl-selenomethionine; these included impregnation of plate by chiral selector and its use as CMPA. Effect of pH, temperature and concentration of chiral selector on enantioresolution is studied. The spots were detected with ninhydrin and iodine vapors. Optically pure amino acids l-phenylalanine, S-benzyl-l-cysteine, and l-methionine were used to synthesize chiral derivatizing reagents (CDRs) based on fluorodinitrobenzene; these CDRs were used to synthesize diastereomers of dl-selenomethionine via nucleophilic substitution of the remaining fluorine atom in these reagents under microwave irradiation for 55 s at 75% (of 800 W) and also by stirring for 50 min at 45 °C. The diastereomers were separated by reversed-phase high-performance liquid chromatography on a C18 column with detection at 340 nm using aq. trifluoroacetic acid and acetonitrile as mobile phase under gradient elution while aq. triethylamine buffer and acetonitrile was successful as mobile phase for separation of diastereomers by reversed-phase thin layer chromatography. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification.

Adherent state apoptosis assay (ASA): a fast and reliable method to detect apoptosis in adherent cells by Rahman Emamzadeh; Mahboobeh Nazari; Somayeh Najafzadeh (4199-4204).
Apoptosis is a tightly controlled biochemical process for cell death. Although the induction of apoptosis is an important mechanism for the screening of many valuable products such as drugs, due to the false signals that usually occur during the isolation of cells from the surface of the culture plate, techniques have limitations in measuring apoptosis in adherent cells. In this study, we investigated the use of RLuc/Annexin V, a probe obtained by the fusion of Renilla luciferase (RLuc) with Annexin V and bound to phosphatidylserine (PS) on the surface of suspended apoptotic cells, as a potentially luminescent probe to assay apoptosis in adherent cells such as Chinese Hamster Ovary (CHO) cells. The probe was overexpressed in Escherichia coli BL21 (DE3) and purified by immobilized metal ion chromatography. The probe assayed for detection of apoptosis in CHO cells. The results show that RLuc/Annexin V binds to the CHO cells with no additional treatment for cell suspension, and the signal of RLuc can be detected by a luminometer. The new assay based on RLuc/Annexin V was named as adherent state apoptosis assay (ASA). This may be a new method for studying apoptosis in adherent cells in a rapid, reliable, and non-invasive way.

A sequential solid phase microextraction (SPME) system consisting of two monolithic capillary columns was developed for simultaneous separation and preconcentration of inorganic arsenic, followed by inductively coupled plasma mass spectrometry (ICP-MS) detection. The N-(β-aminoethyl)-γ-aminopropyltriethoxysilane (AEAPTES) incorporated organic–inorganic hybrid monolithic column was prepared in situ by sol–gel technology in a fused capillary and employed as the extraction medium for SPME in this system, because the amino active sites on the synthesized monolith possess a high adsorption selectivity for As(v). With the on-line design of dual columns and an oxidation coil, As(v) is quantitatively extracted by the first column, and As(iii) in the effluent can be quantitatively extracted by the second column after oxidization to As(v) by a make-up KMnO4 solution. The retained As(v) or As(iii) is then sequentially eluted by diluted HNO3 and introduced to ICP-MS for determination. In this work, the parameters effecting the retention and elution of As(v) and As(iii) were optimized in detail. On-line SPME of 1 mL sample solution gave a signal enhancement factor of 60 for both As(v) and As(iii) using the system. The precision (RSD) for six replicate measurements of 1 μg L−1 As(v) and As(iii) were 3.8% and 3.2%, respectively. The limits of detection (LODs, defined as three times the signal-to-noise ratio) for As(v) and As(iii) were 0.005 μg L−1. The developed method was successfully applied to the speciation analysis of inorganic arsenic in drinking and environmental waters with satisfactory recoveries.

Discrimination and quantification analysis of Acorus calamus L. and Acorus tatarinowii Schott with near-infrared reflection spectroscopy by Xuhui Ying; Yu Pei; Mingying Liu; Guoyu Ding; Min Jiang; Qionglin Liang; Yiming Wang; Gang Bai; Guoan Luo (4212-4218).
A rapid near-infrared reflection (NIR) spectroscopy analysis method was developed for discrimination of the dried rhizome part of Acorus calamus L. and Acorus tatarinowii Schott, two kinds of traditional Chinese herbs that are sometimes mixed or used interchangeably, and the simultaneous determination of their main components β-asarone and α-asarone. The NIR spectra of 25 Acorus calamus L. samples and 25 Acorus tatarinowii Schott samples were collected in integrating-sphere diffused reflection mode and pre-processed with different methods. Principal component analysis (PCA) and discriminant partial least squares (DPLS) were applied to discriminate Acorus calamus L. from Acorus tatarinowii Schott, and the latter method proved better, more visual and effective. The quantitative models of β-asarone and α-asarone were developed using partial least squares regression (PLSR) as multivariate regression method with optimum spectral pre-processing method, wavenumber range and latent variables (LV) numbers, and the results from ultra performance liquid chromatography (UPLC) analysis were taken as reference values. The correlation coefficients of the quantitative models of β-asarone and α-asarone are all above 0.98 while the root mean square errors of prediction (RMSEP) are all below 0.6%, indicating that the models we established have good predictive ability. The results demonstrate that NIR spectroscopy could be used to solve analogous problems for the safety of clinical medication, and can also be applied in the medical industry for the quality control of Acorus calamus L. and Acorus tatarinowii Schott.

Chondroitin sulfate (CS) is an example of a type of acidic mucopolysaccharides, which consist of repeating disaccharide units of glucuronic acid and galactosamine. Since CS has no UV chromophore it is usually detected by its terminal absorption at a wavelength of 200 nm due to the N-acetyl function, resulting in lower sensitivity. Raman spectroscopy (Raman) and near-infrared spectroscopy (NIR) coupled with partial least squares (PLS) can provide rapid, simple, reproducible and non-destructive quantitative analysis of CS, and no sample pre-treatment or pre-separation are required. In the present study we predicted the CS content of tablets using Raman and NIR combined with PLS. Our results showed that the predicted values obtained by NIR were in good agreement with the actual values, with a correlation coefficient of 0.994. In Raman spectroscopy studies, when the CS content of tablets was within the range 7–39% the correlation coefficient and root mean square error of calibration (RMSEC) were 0.998 and 0.578, respectively. When the CS content of tablets was within the range 41–67% the correlation coefficient and RMSEC were 0.994 and 0.742, respectively. High accuracy could thus be achieved within a wider concentration range by use of NIR, whereas relatively high accuracy could be obtained only within a limited concentration range by the use of Raman spectroscopy.

Structure-specific hapten design for the screening of highly sensitive and specific monoclonal antibody to salbutamol by Liqiang Liu; Huang Kuang; Chifang Peng; Libing Wang; Chuanlai Xu (4228-4233).
Salbutamol (SAL) is a short-acting β2-adrenergic receptor agonist (β-agonist) forbidden in livestock production. To develop a more sensitive and specific detection method for SAL, a new hapten was synthesized, keeping the structure of benzene ring side of SAL while changing the tert-butyl group structure that SAL shares with other β-agonists. Ten mice were immunized with a SAL hapten–keyhole limpet hemocyanin (KLH) conjugate, and cells were selected within a urine sample at the cell fusion stage. The best monoclonal antibody (Mab) had an IC50 value of 0.31 ng mL−1 in PBST buffer and an IC50 value of 0.19 ng mL−1 in a swine urine sample diluted five times (with a lower B0 value), and had no cross-reactivity with clenbuterol and other β-agonists. These results show that the hapten design was successful and that the Mab can potentially be used in highly sensitive immunoassays for specifically monitoring the illegal use of SAL in real samples.

Metabolic profiling of Piper species by direct analysis using real time mass spectrometry combined with principal component analysis by Preeti Chandra; Vikas Bajpai; Mukesh Srivastva; K. B. Ramesh Kumar; Brijesh Kumar (4234-4239).
Piper nigrum (black pepper), Piper longum (Indian tipali) and Piper chaba (Bangla tipali) are widely used medicinal herbs in traditional Indian systems of medicine. P. longum as well as P. chaba are considered to be inferior substitutes of P. nigrum. A rapid analytical method for metabolic profiling was developed to generate chemical fingerprints as an alternative means to the only available morphological basis for the identification of these species. Direct analysis using real time mass spectrometry was applied to generate the chemical fingerprints of various parts viz. fruit, leaf and root of these Piper species followed by multivariate analysis for discrimination. Principal component analysis of DARTMS data served as a reliable method for species identification. Phytochemical analysis showed the presence mainly of alkaloids in various parts of Piper species. The results showed that different Piper species could be successfully differentiated using DARTMS fingerprinting together with principal component analysis and this method is promising for the rapid identification and quality control of Piper species.

A CdTe–MPA quantum dot fluorescence enhancement flow method for chlorhexidine determination by João A. C. Barbosa; Vera L. R. G. Abreu; S. Sofia M. Rodrigues; Christian Frigerio; João L. M. Santos (4240-4246).
In this work the fluorescence enhancement that water-soluble CdTe quantum dots (QDs) capped with 3-mercaptopropionic acid (MPA) exhibit in the presence of a biguanide compound, chlorhexidine, was investigated. Acting as an electron-donor ligand, chlorhexidine was able to interact with the defects (mid-gap energy traps) on the QD surface improving the surface passivation and the fluorescence emission. The accomplished fluorescence enhancement was used as the sensing strategy for the implementation of an analytical methodology for the determination of chlorhexidine in pharmaceutical formulations. The developed approach was implemented by resorting to fully automated multipumping flow systems, which improved the versatility and analytical potential provided by QDs thereby enabling some of the shortcomings associated with the commonly used batch procedures to be overcome. Different sized QDs were synthesised and evaluated. A chlorhexidine analytical working range for concentrations between 0.05 × 10−3 and 0.5 × 10−3 mol L−1 was verified with a sampling throughput of about 63 samples per hour. The obtained results were in good agreement with those obtained using the reference method (RD% < ±4.77). A mechanism for the enhancing phenomenon is proposed.

Simultaneous determination of Mo and Ni in wine and soil amendments by HR-CS GF AAS by Wiliam Boschetti; Aline R. Borges; Alvaro T. Duarte; Morgana B. Dessuy; Maria Goreti R. Vale; Jailson B. de Andrade; Bernhard Welz (4247-4256).
The use of high-resolution continuum-source graphite furnace atomic absorption spectrometry (HR-CS GF AAS), equipped with a linear charge-coupled device (CCD) array detector, makes simultaneous determination of more than one element possible. In this work, HR-CS GF AAS was used for the simultaneous determination of Mo (313.259 nm) and Ni (313.410 nm), for which two analytical methods were developed: direct solid sample analysis for soil amendments and direct sample injection for wine samples. For both these methods, a pyrolysis temperature of 1200 °C and an atomization temperature of 2650 °C were used. Aqueous standard solutions were used for calibration. The linear correlation coefficient was higher than 0.997 for the two analytes. Detection limits of 0.05 and 0.8 μg L−1 for wine samples and 0.04 and 0.60 mg kg−1 for soil amendments were found for Mo and Ni, respectively. To investigate the accuracy of the developed method, digested and undigested wine samples were evaluated with spike recovery values between 94% and 106%. For solid samples, three CRM were evaluated, and the values found for Mo were not significantly different from the certified ones; however, those for Ni were always too high. It was found that this was due to a direct line overlap of the Ni line with the Fe line. This effect was overcome by determining Fe using the unresolved analytical line doublet at 312.565/312.568 nm and subtracting this value from the total concentration (Ni + Fe) determined at 313.410 nm. Note that this interference was not observed in wine samples because of their low Fe concentration.

Ultrasonication-assisted rapid determination of epoxide values in polymer mixtures containing epoxy resin by Zhipeng He; Yue Wang; Tingting Zhao; Zichuan Ye; He Huang (4257-4261).
A relatively long time is required to determine the epoxide value of an epoxy resin or epoxy compound by chemical analysis (titration) methods, including the commonly used hydrochloric acid–acetone method, which may be referred to as the hydrochloric acid–acetone-standing method, because the mixture of epoxy resins or epoxy compounds, hydrochloric acid and acetone must be left standing in the dark for at least 30 min. In this study, ultrasonication was first introduced to this method, aiming to rapidly determine the epoxide value of an epoxy resin by accelerating the dissolution of epoxy resin in acetone solvent as well as the reaction between epoxide groups and hydrogen chloride. Further study of this approach was then carried out to determine the epoxide values of a series of epoxy resin–polyethylene glycol mixtures. This new approach which may be referred to as the hydrochloric acid–acetone-ultrasonication method provides a much faster and more accurate measurement of the epoxide value of an epoxy resin (within 2 min) or its mixture with polyethylene glycol (PEG-4000, within 5 min), compared to the hydrochloric acid–acetone-standing method. This ultrasonication method may be readily extended to other chemical analysis (titration) methods.

A simple, rapid and sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) assay method has been developed and validated for the determination of anagrelide in human plasma samples using nevirapine as internal standard (IS). The analyte and the IS were extracted from 100 μL of human plasma using solid-phase extraction with no drying, evaporation and reconstitution steps. The chromatographic separation was achieved on a C18 column by using a mixture of methanol and 0.1% formic acid in 5 mM ammonium acetate (80 : 20, v/v) as the mobile phase at a flow rate of 1.0 mL min−1. The linearity of the method was established in the concentration range 0.05–10.0 ng mL−1 with r2≥ 0.99. Method validation was performed as per FDA and EMEA guidelines and the results met the acceptance criteria. Data acquisition was done by multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer in the positive ionization mode. The intra-day and inter-day precision (%CV) and accuracy results in five validation batches across five concentration levels were well within the acceptance limits. The validated method was successfully applied to a pharmacokinetic study in healthy South Indian male subjects under fasting conditions with 0.5 mg anagrelide capsules.

Herein, we have demonstrated the simultaneous and selective electrochemical determination of dihydroxybenzene isomers at a reduced graphene oxide (RGO) and copper nanoparticles (Cu-NPs) composite modified electrode. The RGO/Cu-NPs composite was prepared by a single-step electrochemical reduction method. The synthesized RGO/Cu-NPs composite was characterized using scanning electron microscopy and elemental analysis. Linear sweep voltammetry was employed for the simultaneous determination of hydroquinone (HQ), catechol (CC) and resorcinol (RC). A well defined and more enhanced oxidation peak response is observed for HQ, CC and RC at the RGO/Cu-NPs composite electrode compared to other modified electrodes, which indicates fast electron transfer from dihydroxybenzene isomers at the RGO/Cu-NPs composite electrode. The composite modified electrode shows high electrocatalytic activity towards the oxidation of HQ, CC and RC. The electrochemical sensor shows a wide linear response in the concentration range of 3 μM to 350 μM, 3 to 350 μM and 12 μM to 200 μM for HQ, CC and RC respectively with a detection limit of 0.032 μM, 0.025 μM and 0.088 μM (S/N = 3). In addition, the proposed sensor shows good selectivity and stability along with good precision and consistency. The obtained results clearly demonstrate that the RGO/Cu-NPs composite can be an advanced electrode material for the real time sensing of dihydroxybenzene isomers.

Ginger (Zingiber officinale) headspace has been extracted using a novel polyaniline–nylon-6 (PANI–N6) nanocomposite fabricated by electrospinning. GC-MS (gas chromatography-mass spectrometry) and chemometrics have been used to identify the components of the fragrance. Chemometric resolution techniques were utilized to improve the resolution, qualitative and quantitative determination of co-eluted compounds in GC-MS. In this way, chromatographic problems such as baseline/background contribution, low S/N peaks and co-elution occurring during chromatographic analysis were solved. Moreover, principal component analysis was used to determine hidden structures and to identify those volatiles which were most differentiated between the fragrance and the essential oil of ginger. The results show that the fragrance and the essential oil of ginger are different enough in terms of chemical composition to put them into two distinct classes using 17 components which account for most of the variation. Among the 62 identified components of ginger fragrance, α-phellandrene (18.14%), α-zingiberene (16.45%), (E,E)-α-farnesene (7.21%), camphene (5.47%) and geranial (4.38%) are the major components. The results proved that the present procedure may be useful for the comprehensive analysis of complex natural aromas such as ginger fragrance.

Determination of 187 pesticide residues in edible fungi by liquid chromatography-tandem mass spectrometry by Qiaoying Chang; Chunlin Fan; Hui Chen; Jian Kang; Minglin Wang; Guofang Pang (4288-4304).
A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is developed for simultaneous determination of 187 pesticide residues in edible fungi. Samples of edible fungi (20 g) were homogeneously extracted with acetonitrile, the extracts were cleaned up using stacked solid-phase extraction (SPE) cartridges containing graphitized carbon black and aminopropyl sorbents. The residue pesticides were eluted with acetonitrile–toluene (3 : 1, v/v), and the eluates were concentrated and used for analysis. In the linear range of each pesticide, the linear correlation coefficient R2 was equal to or greater than 0.990. At low and high fortification levels, recoveries ranged from 70% to 118%. The relative standard deviation (RSD) was always below 30% and was below 25% for 169 pesticides, accounting for 90%. The limit of detection (LOD) was 0.01–85 μg kg−1, 165 pesticides had LOD ≤10 μg kg−1, accounting for 88%. The proposed method is suitable for determination of 187 pesticide residues in shiitake, black fungus, nameko and enoki mushroom.

The application of Fourier transform infrared (FT-IR) spectroscopy for in-line monitoring of the synthesis process of 4-amino-3,5-dimethyl pyrazole is described. The obtained data matrix of IR was analyzed by a well known chemometric method, independent component analysis (ICA), which determined the concentration profiles and the spectra of the reactant, intermediates and product. The geometric configurations of the intermediates were fully optimized and the vibrational frequencies computed at the density functional theory (DFT) B3LYP/6-31G level of theory. The computational results by the ICA method are in good agreement with those by the quantum chemical calculation method, which demonstrated the reliability of the proposed ICA method. The possible synthesis mechanism was deduced, and the results indicated that ICA combined with in-line FT-IR spectroscopy can be used to study the synthesis mechanism of 4-amino-3,5-dimethyl pyrazole successfully.

Zhi-Zi-Da-Huang decoction (ZZDHD) is an effective formula to treat alcoholic liver disease in China for nearly 2000 years. However, compounds in the formula are still not clear due to the complexity of the coexisting constituents. In this study, a sensitive integrated method based on HPLC-DAD-ESI-MS (TOF) and HPLC-DAD-ESI-MS/MS (QqQ) was developed and 85 chemical compounds in ZZDHD were identified. By TOF/MS, the quasi-molecular ions of the constituents were deduced according to the accurate mass of corresponding positive and negative ions, and product ion scan was then carried out using QqQ/MS. The fragments of each compound were observed by TOF/MS and/or QqQ/MS. Compounds with different chemical structures and concentration levels were tentatively identified according to their chromatographic retention time, UV absorption and MS data. Fragmentation patterns of six types were proposed to verify the identification results. It was found that at TOF/MS mode, a retro diels-alder (RDA) reaction occurred at bonds 1 and 3 in C-ring of flavanone 7-O-diglycosides when 4′-OH exists at B-ring. While the reaction was not observed if a methoxyl group appeared at the same site of flavanone skeleton. Another important phenomenon was noticed that 3-sinapoyl-5-caffeoylquinic acid, 4-sinapoyl-3-caffeoylquinic acid, and sinapylglucoside were detected only in ZZDHD while they were not found in four component herb extracts. The phenomenon revealed that changes of compounds happened when the component herbs were decocted together. The results in the paper are favorable of the clinic dosage and have laid a foundation for the subsequent research of ZZDHD in vivo.

A simple and efficient HPLC method is developed for the fingerprint analysis of Flos Sophorae Immaturus and for the simultaneous determination of three flavonoids in Flos Sophorae Immaturus, namely rutin, narcissin and quercetin. The separation of analytes is conducted on a Dikma Diamonsil C18 column (250 mm × 4.6 mm, 5 μm) at 35 °C; the wavelength of UV detector was set at 254 nm; the mobile phases were composed of acetonitrile and aqueous acetic acid (0.5%, v/v). A gradient elution was carried out at a flow rate of 1.0 mL min−1. A common chromatographic fingerprint consisting of 9 characteristic peaks is established among 14 batches of Flos Sophorae Immaturus samples, which were collected from different source areas of China. The constituents in Flos Sophorae Immaturus were further identified by HPLC-DAD-ESI-MS/MS and most of them were flavonoids. Multiple chemometrics analysis, including similarity analysis (SA), hierarchical cluster analysis (HCA) and principal component analysis (PCA) were performed to classify samples. In quantitative analysis, all of the calibration curves showed good linear regression (R2≥ 0.9991) within the tested ranges, and the mean recoveries ranged from 100.28% to 101.08%. Three flavonoid compounds in Flos Sophorae Immaturus were simultaneously quantified by the established method. The results demonstrate that the developed method is accurate and effective, and could be readily utilized for the comprehensive quality control of Flos Sophorae Immaturus.

A rapid, sensitive and selective method for determining phthalanilic acid residue in bean, fruits and vegetables based on a modified quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction procedure was developed by ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS). The samples were extracted by acidified acetonitrile (0.4% (v/v) formic acid) and simultaneous liquid–liquid partitioning formed by adding anhydrous magnesium sulfate (MgSO4) and sodium chloride (NaCl). The extract was then cleaned up by dispersive-SPE using florisil and graphitized carbon black (GCB) as selective sorbent. Parameters including the matrix effect, linearity, precision, accuracy and stability were undertaken. The method showed good linearity in the concentration range of 10–1000 μg L−1, with correlation coefficients ≥0.997. Recovery studies were performed at three fortification levels of 10, 100 and 1000 μg kg−1 in soybean, apple, grape, cucumber, tomato and pepper. Further optimization of sample preparation and determination achieved recoveries ranging between 70.1% and 105.3% for the analyte with 0.7–14.9% intra-day relative standard deviations (RSD) and 0.2–8% inter-day RSD at three spiked levels (10, 100 and 1000 μg kg−1). The limits of detection (LOD) were below 1 μg kg−1, and the limits of quantification (LOQ) did not exceed 3 μg kg−1. The effects of two columns on the separation were also investigated. The method is demonstrated to be convenient and reliable for determination of phthalanilic acid in bean, fruits and vegetables. With the developed method, 60 samples of commercial fruit products (grape, apple), soybean and vegetables (pepper, tomato, cucumber) were analyzed. Phthalanilic acid was not detected in all samples.

In weakly acidic and neutral media, palladium(ii) or sodium dodecyl benzene sulfonate (SDBS) can separately quench the fluorescence of quinolone antibiotics (FLQs) in varying degrees. When Pd(ii) reacts with SDBS and FLQs to form ternary complexes, enhanced fluorescence quenching of FLQs can be observed. This synergistic fluorescence quenching effect has high sensitivity for Pd(ii) detection, and the detection limit could reach 0.13 ng mL−1. Based on this, a rapid, simple and reliable method for the determination of Pd(ii) in aqueous samples was established. The optimum reaction conditions of the method were tested. The reaction information of the FLQs–SDBS–Pd(ii) system was investigated by absorption spectra and fluorescence spectra, and quantum chemical calculations using density functional theory B3LYP under a polarizable continuum model (PCM). The results showed that the pefloxacin (PEF) molecule exists as a zwitter-ion of HL±, reacting with Pd(ii) to form plane bis(PEF)Pd chelates, which further bind with two SDBS molecules to form ternary complexes. The composition of Pd(ii)–PEF–SDBS complex was found to be 1 : 2 : 2. The ternary complexes resulted in a higher fluorescence quenching efficiency and enhanced the sensitivity for the determination of Pd(ii).

Determination of total glutathione in earthworms by ultra-high performance liquid chromatography with fluorescence detection by Juan José Berzas Nevado; Rosa Carmen Rodríguez Martín-Doimeadiós; Francisco Javier Guzmán Bernardo; Carolina Rodríguez Álvarez (4353-4359).
A new, fast and simple methodology for the determination of total glutathione in earthworms, excellent bioindicators of oxidative stress, using ultra-high performance liquid chromatography with fluorescence detection is presented. Total glutathione could be a biomarker of oxidative stress caused by heavy metals such as mercury. The strategy for the analysis was to transform oxidized glutathione (GSSG) into reduced glutathione (GSH) and then to derivatize total GSH with o-pthalaldehyde to a fluorescent adduct. The analysis was carried out using an Eclipse XDB-C18 (50 mm × 4.6 mm × 1.8 μm) column at 25 °C, and using 100 mmol L−1 phosphate buffer at pH 7.0 and acetonitrile (phosphate buffer–acetonitrile, 85 : 15, v/v) as the mobile phase at 1.5 mL min−1. The volume injected was 5 μL and the excitation and emission wavelengths were 340 and 420 nm, respectively. Under the optimized conditions, the retention time of the derivatized analyte was as short as 0.536 min, which allows also a very short time between consecutive injections. The instrumental limits of detection and quantification were 1.8 and 5.3 μg L−1 (5.7 and 17.0 nmol L−1) and the calibration curve was linear up to 5 mg L−1 (16.3 μmol L−1). The methodology was also proved to be robust and precise. The methodology was validated using earthworms spiked with GSH, obtaining recoveries close to 100%.

In this paper, a moving interaction boundary (MIB) method was developed using a guest compound and a host molecule of cyclodextrin (CD) in capillary electrophoresis (CE). With norfloxacin (Nor) as the model guest compound, experiments were carried out and the results revealed that (1) the MIB method can be designed as a tool for focusing target guest molecules in complex biological samples (e.g., urine); (2) the focusing is highly selective; (3) compared with capillary zone electrophoresis (CZE), a 143-fold sensitivity increase, in terms of the peak height, can be achieved and (4) moreover, the actual application of this MIB-based focusing method for the analysis of a complex biological sample (e.g. urine spiked with Nor), containing numerous metabolites, was carried out. The concept of MIB and its selective focusing has potential significance toward host–guest interaction and separation science.

Herein, a gold nanoparticle (GNP)-based colorimetric aptasensor has been developed for detecting adenosine with exonuclease III (Exo III) assisted recycling amplification. In this aptasensor, two kinds of single-stranded oligonucleotide functionalized GNP probes (Oligo 1-GNPs and Oligo 2-GNPs) are firstly hybridized with one complementary single-stranded oligonucleotide (linker) with the motif of adenosine aptamer, resulting in the formation of GNP aggregates (cross-linked GNPs). In the presence of adenosine, the linker would form a double-stranded DNA (dsDNA) with a recessed 3′-terminus due to its structural switching property, which would lead to disassembly of the cross-linked GNPs. However, upon the addition of Exo III, the dsDNA is enzymatically digested from the 3′-hydroxyl termini, liberating the adenosine. The released adenosine can then interact with another linker and a new round of enzymatic digestion is started. Therefore, remarkable signal amplification is achieved because the disassembly of cross-linked GNPs is strongly dependent on the free adenosine in the reaction mixture. The experimental results demonstrate that the Exo III-assisted GNP-based colorimetric aptasensor is more sensitive (ca. 10 times) than that without Exo III amplification. The Exo III-assisted GNP-based colorimetric aptasensor detects adenosine downs to 5.6 nM, with a linear relationship from 10 nM to 1 mM. Moreover, the Exo III-assisted GNP-based colorimetric aptasensor has been successfully applied to detect Jinshuibao capsule (JSBC) containing adenosine in both a buffer solution and a diluted human serum, demonstrating that this assay has great potential to be employed for detecting adenosine in real samples.

Simultaneous determination of nine major constituents in Agrimonia pilosa Ledeb. by HPLC-DAD-ESI-MS/MS by Qinghua Jiang; Jianghao Ma; Ying Wang; Liqing Ding; Lixia Chen; Feng Qiu (4373-4379).
Herba Agrimoniae is a traditional Chinese medicine. To date, there is no quantitative control method for Agrimonia pilosa (A. pilosa) with main active constituent contents listed in the Chinese Pharmacopeia. A high performance liquid chromatography coupled with diode-array detection and electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS/MS) method is established for the characterization and simultaneous quantification of nine major constituents in A. pilosa. Furthermore, the contents of these constituents of A. pilosa from 21 different regions of China were analyzed and compared. All calibration curves show good linearity (r≥ 0.999). The HPLC-DAD method shows good precision, repeatability and stability for the quantification of the nine constituents in A. pilosa with the standard deviations below 3.29%. The recoveries for the quantified constituents are within the range of 95.44–102.55%. The qualitative and quantitative method was proved to be sensitive, accurate and reliable to determine the constituents in A. pilosa. The method is successfully applied to differentiate the A. pilosa of different regions.

The derivatization of metabolites is inevitable for GC-MS based global metabolic profiling. This article reports a GC-MS-based protocol using methoximation followed by silylation with BSTFA + 1%TMCS for the analysis of urine metabolites. The protocol has been thoroughly developed and optimized from derivatization to detection. The obtained chromatograms were much cleaner due to the absence of multi-peaks of sugars, such as glucose. Validation was performed with chemical standards and urine samples and proved that the methodology is efficient, rapid and reliable with linear responses, low detection limits and good precision and recovery. The method was successfully applied in the characterization of the metabolic phenotype of type 2 diabetic KK-Ay mice. Partial least squares-discriminant analysis (PLS-DA) and t-test analysis illustrated that there were seven metabolites (glyceric acid, hippuric acid, glucose, sorbitol, galactonic acid, myo-inositol, turanose) having distinct differences between normal C57BL/6J and type 2 diabetic KK-Ay mice.

Simultaneous HPLC quantification of seven chromones in Aloe barbadensis Miller using a single reference standard by Jia-Sheng Zhong; Jin-Zhi Wan; Yu-Peng Liu; Wen-Jing Ding; Xiao-Fang Wu; Zhi-Yong Xie (4388-4395).
Multi-component analysis has been considered to be one of the key techniques for quality control of traditional Chinese medicines. However, the shortage and high cost of reference standards have become the greatest obstacles in implementing this goal. An alternative method using only an easily available reference standard as the internal standard to determine multiple analogues has gained increasing attention. In the present study, a practical HPLC method was established for the simultaneous quantification of seven chromones in Aloe barbadensis Miller using a single reference standard. An easily available compound in the plant named 8-C-glucosyl-7-O-methyl-(S)-aloesol (3) was selected as the internal standard, and the other six chromones were determined by their relative response factors. The results indicated that the established method was accurate and reliable for the routine quality control of A. barbadensis. 6 batches of A. barbadensis samples were analyzed using this method and no significant difference was observed in the results obtained using the established method and external standard method. The newly developed method can serve as an alternative approach when reference standards are difficult to obtain. This achievement would promote the quality control of A. barbadensis.

Precise and accurate porosity measurement is essential for characterization of porous materials. Considering Archimedes' principle based liquid displacement methods of measuring porosity we have developed an excellent modified micro-volumetric method of porosity measurement. Changes in the liquid level in a glass pipette after immersing and also removing the porous sample were recorded by using a digital camera and analysed by ImageJ® software. Results of porosity measurement through a micro-volumetric method were compared with micro-CT results. Bland–Altman analysis showed a much higher precision and accuracy for our micro-volumetric method (bias = −0.023, CI: ]−0.459, 0.413[, SD = 1.96 Ơ) compared to the micro-CT method (bias = 6.075, CI: ]−20.993, 33.142[, SD = 1.96 Ơ). Our highly precise and accurate micro-volumetric method of porosity measurement is particularly applicable to small ultra-light highly porous materials.

Baseline correction for Raman spectra using an improved asymmetric least squares method by Shixuan He; Wei Zhang; Lijuan Liu; Yu Huang; Jiming He; Wanyi Xie; Peng Wu; Chunlei Du (4402-4407).
Baseline shifts exist in many types of Raman spectrometers. Acquired spectra normally contain the desired signals as well as undesirable elements such as background noise. In this paper, an improved asymmetric least squares (IAsLS) method has been proposed for the baseline correction of Raman spectra. The baseline correction algorithm is initiated by the raw spectrum baseline, and this baseline can be estimated using a polynomial fitting method. For the simulated Raman spectra, the performance of the proposed algorithm was evaluated and compared with the asymmetric least squares (AsLS) method and Jiang's method. The results showed that it is improved by sixteen fold and nine fold respectively. This proposed IAsLS method was successfully applied to practical Raman spectral data and the results in the paper indicate that the baseline of Raman spectra can be automatically subtracted.

A simple and rapid method for chiral separation of amlodipine using dual chiral mobile phase additives by Jiaqi Xie; Qi Tan; Lin Yang; Shenzhi Lai; Shaotan Tang; Changqun Cai; Xiaoming Chen (4408-4413).
A simple, rapid and sensitive method for chiral separation of the amlodipine racemate was developed. (R)- and (S)-amlodipine enantiomers were separated by achiral HPLC, utilising dual chiral mobile phase additives, within 16 min, which was much faster than the previous similar studies. The method was established on an achiral WondaSil C18 column, using methanol–water (45 : 55 (v/v), pH 2.5) containing 7.5 mmol L−1 sulfobutylether-β-cyclodextrin (SBE-β-CD) and 0.3 mmol L−1 polyethylene glycol-20 000 (PEG-20M) as the mobile phase. Effects of additives and concentration, content of methanol and pH on the enantioseparation were investigated. The method was validated for linearity, repeatability, limits of detection (LOD) and limits of quantification (LOQ). The calibration curve (R(S)2 = 0.9998) was constructed in the range of 10–500 μg mL−1 for (S)-amlodipine. Repeatability (n = 6) showed less than 2% relative standard deviation (R.S.D.). LOD and LOQ were found to be 0.032 and 0.106 μg mL−1 for (S)-amlodipine. This method was flexible, simple and economically advantageous over the use of a chiral stationary phase, and was successfully applied to the determination of the amlodipine enantiomers in a commercial pharmaceutical tablets study.

Development of a direct competitive chemiluminescent ELISA for the detection of nitrofurantoin metabolite 1-amino-hydantoin in fish and honey by Quan Wang; Ying-Chun Liu; Yong-Jun Chen; Wei Jiang; Jin-Lei Shi; Yan Xiao; Meng Zhang (4414-4420).
A direct competitive enzyme-linked immunosorbent assay (ELISA) with chemiluminescent (dcCLELISA) detection for 1-amino-hydantoin (AHD) was developed in this study. AHD was derivatised with 4-carboxybenzaldehyde to produce 1-[(4-carbo-benzylidene)-amino]-imidazolidin-2,4-dione (CPAHD). Monoclonal antibodies (MAb) against AHD were prepared through immunization of BALB/c mice with synthesized CPAHD–Jeffamine–BSA as an antigen. Luminol, p-iodophenol, and urea peroxide mixture solution served as the substrate in CLELISA. The specificity of the MAb, estimated as the cross-reactivity values from the dcCLEILISA assay for 1-[(4-nitro-benzylidene)-amino]-imidazolidin-2,4-dione (NPAHD) and CPAHD, was 100% and 39.67%, respectively. All other compounds showed less than 0.01%. The sensitivity of the antibody, estimated as the IC50 value, was 0.60 μg L−1. The limits of detection for dcCLELISA in fish and honey samples were 0.1 and 0.28 μg kg−1, respectively, and the mean recovery values ranged from 83.6% to 94.7% for fortified samples at levels of 0.25–10 μg kg−1 with coefficient of variation values below 15%. Finally, dcCLELISA was compared to a commercial kit in the detection of AHD in spiked fish and honey samples. The immunoassay method described here showed a broad detection range and high sensitivity. It could be used for high-throughput monitoring of AHD in fish and honey samples and possibly other types of food.

Simultaneous extraction of anthracyclines from urine using water-compatible magnetic nanoparticles with a dummy template coupled with high performance liquid chromatography by Wenyue Zou; Pierre Dramou; Lien Ai Pham-Huy; Kai Zhang; Jia He; Chuong Pham-Huy; Deli Xiao; Hua He (4421-4429).
Water-compatible magnetic molecularly imprinted polymers (M-MIPs) for extraction and pre-concentration of anthracyclines (ANTs) from urine have been successfully synthesized by a non-covalent method using epirubicin (EPI) as a dummy template, methacrylamide as a functional monomer, and ethylene glycol dimethacrylate as a cross-linker. The obtained M-MIPs were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and vibrating sample magnetometer (VSM). Adsorption kinetic and isotherm studies were carried out, which indicated that the M-MIPs displayed a rapid dynamic process and a high adsorption capacity. The adsorption behavior was discussed in detail, showing that it could be described as a chemisorption process and both external surface diffusion and intra-particle diffusion contributed to the adsorption mechanism. Furthermore, the binding sites were found to be heterogeneous for M-MIPs, while homogeneous for M-NIPs. The selectivity of M-MIPs demonstrated higher affinity for target EPI and EPI-analogues over other structurally unrelated compounds. A rapid solid-phase extraction (SPE) method using M-MIPs as sorbents coupled with high performance liquid chromatography (HPLC) was established for simultaneous determination of ANTs in urine samples. The recoveries ranged from 93.9% ± 5.2% to 100.0% ± 3.4% with the limit of detection of 0.6–2.4 ng mL−1. Moreover, the M-MIPs could be regenerated, which could be utilized for several cycles with no obvious decrease in the adsorption capacity. The results indicated that the proposed method is a practical approach for simultaneous determination of ANTs in urine.

Fluorescence immunoassay based on carbon dots as labels for the detection of human immunoglobulin G by Lei Zhu; Xin Cui; Jing Wu; Zhenni Wang; Peiyao Wang; Yu Hou; Mei Yang (4430-4436).
A rapid, selective and environmentally friendly fluorescence immunoassay strategy based on carbon dots (CDs) is developed for the detection of human immunoglobulin G (IgG, antigen). Firstly, in this protocol, CDs with high quantum yield were synthesized by hydrothermal process using citric acid as a carbon source and then carboxylated by bath sonication with NaOH and ClCH2COONa. Subsequently, carboxyl-functionalized CDs were conjugated with goat antihuman IgG (gIgG, antibody) using an EDC–NHS amidization method to obtain the CDs-labeled gIgG, and this obtained conjugate CDs–gIgG was then incubated with a limited amount of human IgG for immunoreaction between antigen and antibody. The immunocomplex formed on the surface of carboxylated CDs resulted in the increase of fluorescence intensity. Under the optimal conditions, a linear relationship between fluorescence change ratio (F−F0)/F0 and human IgG concentration in a range from 0.05 to 2.0 μg mL−1 was obtained with a detection limit of 0.01 μg mL−1. This method has been successfully applied to the determination of IgG in human serum, with recoveries in the range of 95.8–111.5%. The results indicate the great promise for CDs as labels in fluorescence immunoassays.

Stability-indicating LC method for the determination of cephalothin in lyophilized powder for injection by Karen de Souza Rugani; Hérida Regina Nunes Salgado (4437-4445).
A stability-indicating gradient reversed phase liquid chromatography (RP-LC) method has been developed for the quantitative determination of cephalothin (CET), an antimicrobial compound, in the presence of its impurities and degradation products generated from forced degradation studies. The developed method is also applicable for related substance determination in bulk drugs. The chromatographic separation was achieved on an Agilent Eclipse XDB-Phenyl, 250 mm × 4.6 mm, 5 μm column with a mobile phase containing a gradient mixture of solutions A (aqueous ammonium phosphate buffer, pH 4.5) and B (acetonitrile). The flow rate was 1.0 mL min−1 and the detection wavelength was 238 nm. The drug substance was subjected to stress conditions of hydrolysis, oxidation, photolysis, humidity and thermal degradation. Considerable degradation was found to occur in basic, acidic and oxidative stress conditions. In the developed high performance liquid chromatography (HPLC) method, the resolution between CET and its potential degradation products was found to be greater than 2.4. Further, the peak purity of CET in all conditions was more than 99% and proved the stability-indicating power of this method. The less active metabolite of cephalothin, deacetylcephalothin (impurity B), was identified and showed significant formation especially in basic conditions. This method is capable of detecting the degradation products of CET at a level of 0.05% with respect to the test concentration of 500 μg mL−1 for a 10 μL injection volume. The developed HPLC method was validated with respect to linearity, accuracy, precision, specificity, limit of quantitation, limit of detection and robustness. To our knowledge, a rapid stability-indicating LC method for CET has not been published elsewhere and this method can be applicable to the evaluation of the quality of product samples as well as in stability studies of cephalothin.

In the present study, a carbon paper electrode (CPE)/cells-in-paper/CPE sandwich device was developed for the in situ detection of small molecules produced from cells growing in paper 3 dimensional matrix. To demonstrate the real-time assay capability of the electrodes/cells-in-paper sandwich device, carbon nanotube/graphene/MnO2 nanocomposite was synthesized to functionalize the working electrode. Scanning electron microscopy, transmission electron microscope and X-ray photoelectron spectroscopy characterization proved that MnO2 nanoparticles were uniformly distributed on nanotube sidewalls and graphene sheets. The carbon nanotube/graphene/MnO2 nanocomposite functionalized electrode/cells-in-paper sandwich device showed specific response against hydrogen peroxide. The fully assembled device displayed a linear range up to 25 mM with a sensitivity of 6.25 μA mM−1 cm−2 and a detection limit of 6.7 μM hydrogen peroxide. In addition, in situ detection of hydrogen peroxide production from cells growing in matrigel impregnated paper was successfully demonstrated on the electrodes/cells-in-paper sandwich device, highlighting the potential application of this low-cost paper analytical device for cell biology studies.

A simple, rapid and effective extraction method based on matrix solid phase dispersion (MSPD) and ultrafast liquid chromatographic (UFLC) was developed and validated for the simultaneous cleaning-up and quantitative extraction of illegal dyes (Sudan I–IV) from the roots of Salvia miltiorrhiza Bunge. The experiment parameters, such as dispersing sorbent, the ratio of sorbent–sample, washing solvent and elution solvent were evaluated to find the optimal MSPD conditions. The optimal conditions were 0.5 g of Salvia miltiorrhiza Bunge, 1.0 g of silica gel as dispersing sorbent, a volume of 10 mL of water as the washing solvent and 4 mL of acetonitrile–methanol (9 : 1, v/v) as the elution solvent. Under these conditions good linearity for all the Sudan dyes ranged from 0.10 μg g−1 to 10 μg g−1 (r2≥ 0.9992). The recoveries at three spiked levels (0.1, 1.0, 5.0 μg g−1) were between 80.6% and 96.1% with relative deviations (RSDs) ranging from 2.3% to 8.6%. The limits of detection ranged between 0.013 and 0.024 μg g−1 which were twenty times lower than the values required by European regulations. This method has potential to be applied for the determination of illegal dyes in complicated traditional Chinese herb materials.

A chemically modified electrode with hydroquinone derivative based on carbon nanoparticles for simultaneous determination of isoproterenol, uric acid, folic acid and tryptophan by Mohammad Mazloum-Ardakani; Nooshin Rajabzadeh; Afsaneh Dehghani Firouzabadi; Ali Benvidi; Mohammad Abdollahi-Alibeik (4462-4468).
A nanostructure electrochemical sensor with a carbon paste electrode has been used to study the electrocatalytic oxidation of isoproterenol (IP) in the presence of uric acid, folic acid and tryptophan. In this work carbon nanoparticles (CNPs) and 7-(3,4-dihydroxyphenyl)-10,10-dimethyl-9,10,11,12-tetrahydrobenzo[c]acridin-9 (7H)-one (DDTA) were used for investigation of the electrochemical behaviour of IP. An apparent charge transfer rate constant of ks = 1.99 s−1, and transfer coefficient of α = 0.51, for the electron transfer between the modifier and the carbon paste electrode were calculated. In the optimum pH (pH = 9.0), the oxidation of IP at the modified electrode occurs at a potential about 298 mV less positive than the unmodified carbon paste electrode. The catalytic rate constant (k = 134.28 M−1 s−1) and diffusion coefficient (D = 3.7 × 10−5 cm2 s−1) were calculated for IP by chronoamperometry. Differential pulse voltammetry (DPV) exhibited two linear dynamic ranges of 0.25 to 20.0 μM and 20.0 to 2000.0 μM for IP. There was a decrease in sensitivity of the second linear segment due to kinetic limitations. Also, the modified electrode was used to determine IP in the presence of uric acid (UA), folic acid (FA) and tryptophan (TRY) by differential pulse voltammetry. Simultaneous determination of these four compounds was performed on the proposed sensor for the first time. This sensor was used for the determination of IP from an ampoule sample. The detection limit of the IP was calculated as 0.075 μM.

Back cover (4469-4470).