Analytical Methods (v.6, #2)

Front cover (319-319).

Inside front cover (320-320).

Contents list (321-332).

PCR – the polymerase chain reaction by Analytical Methods Committee, AMCTB No 59 (333-336).
DNA-based procedures are becoming increasingly common within the analytical laboratory where the polymerase chain reaction (PCR) has become an indispensable technique. Developed in 1985 by Kary B. Mullis, PCR revolutionized the way that deoxyribonucleic acid (DNA) could be copied. Mullis's invention allowed researchers to make millions of copies of a selected DNA region within hours. Today, PCR can take minutes. PCR has widespread analytical applications in the food, environmental, medical and forensic fields. This Technical Brief covers the basics of PCR and some of its variations.

A new method to determine the thickness of platinum nanofilm simply by measuring its electrical resistance by Yujing Sun; Zhiwei Wen; Fugang Xu; Yue Zhang; Yan Shi; Haichao Dai; Zhuang Li (337-340).
In this report, it was found that the Napierian logarithm of the electrical resistance is proportional to the reciprocal thickness for the platinum nanofilms. A new method was proposed to determine the thickness of platinum nanofilm simply by measuring its electrical resistance, which is fast and cost effective.

Enantioselective chromatographic resolution using a homochiral metal–organic framework in HPLC by Mei Zhang; Xu-Dong Xue; Jun-Hui Zhang; Sheng-Ming Xie; Yan Zhang; Li-Ming Yuan (341-346).
A homochiral MOF was used as a new chiral stationary phase and had an excellent recognition ability for various racemates in HPLC. The results indicated that the optical selectivity of the natural chiral MOF column is practical in HPLC, which promotes the application of chiral MOFs for enantioseparation.

We report the photophysical effects of nitric oxide and S-nitrosocysteine on an antibacterial agent, acridine orange, and describe its use in sequential sensing of NO, cysteine and S-nitrosocysteine in a protein, as well as Hg2+. We show that cysteine can play a major role in controlling NO-induced resistance to bacteria.

Gold nanoparticles were generated and immobilized on the surface of an antibody conjugated with a hydrophilic Fischer carbene complex with oligo-ethyleneglycol tethers, enabling rapid visual detection of the corresponding antigen specifically, even in serum, at a very low concentration without any instrumentation.

Radioactive strontium-90 scattered by a nuclear power plant accident was specifically quantified by conventional inductively coupled plasma quadrupole mass-spectrometry (ICP-QMS) preceded by on-line chelate column separation (based on lab-on-valve) and oxygen reaction (designated the cascade step). The proposed system overcomes the isobaric interference of 90Zr, whose soil concentration exceeds that of 90Sr by more than six orders of magnitude. In addition, the system requires no ultimate mass spectrometry or radioactive 90Sr standards. The radioactive 90Sr standard was replaced with the stable isotope 88Sr as a pseudo-standard. The modified ICP-QMS system yielded a precise, reproducible sharp 90Sr peak in the ICP-MS profile. The elution time of 90Sr was highly reproducible (RSD = 0.5%). After implementing the cascade-step, the detection limit (DL) was 2.3 Bq L−1 (equivalent to 0.46 ppq as 90Sr). Analysis of microwave-digested soil yielded a DL of 3.9 Bq kg−1 (equivalent to 0.77 ppq as 90Sr). The 90Sr from environmental contaminated soil samples collected from areas at a distance of 10 and 20 km from the Fukushima Daiichi nuclear power plant ranged from 52 Bq kg−1 to 73 Bq kg−1, with no statistical difference between the proposed and general methods at 95% confidence level. The proposed method offers an attractive alternative use for ICP-other ionization mass spectrometry as an enrichment or purification step, thereby expanding the scope of ICP-mass-spectrometric analysis.

Screening for mutations in the tumor-suppressor genes BRCA1 and BRCA2 is of great importance for breast and ovarian cancer prevention. We describe a methodology for mutation screening and detection based on acoustic wave devices. In particular, we detect four mutations located in BRCA1 and BRCA2 genes using the quartz crystal microbalance technique. The detection is based on measurements of the acoustic ratio of dissipation versus frequency change (ΔD/ΔF) of double-stranded DNA molecules bound to the device surface that are produced after PCR amplification and restriction digestion; the acoustic ratio has been shown to be a measure of the intrinsic viscosity of the attached molecules, which, in turn, depends on the size of the dsDNAs. Novel features of this approach are the lack of a hybridization step, the label free sensing of the length, rather than mass, of the DNA molecules and the direct detection of the digested DNA products without prior purification. The method is generic, simple and capable of detecting single base mutations to long genomic rearrangements; it is also suitable and applicable to a Lab-on-a-chip concept.

Multianalytical approach to explain the darkening process of hematite pigment in paintings from ancient Pompeii after accelerated weathering experiments by Maite Maguregui; Kepa Castro; Héctor Morillas; Josu Trebolazabala; Ulla Knuutinen; Rita Wiesinger; Manfred Schreiner; Juan Manuel Madariaga (372-378).
In this paper, recently excavated fresco painting fragments from the House of Marcus Lucretius (Pompeii) and not exposed to the atmosphere since the eruption of the Mount Vesuvius were subjected to a controlled SO2 atmosphere and high relative humidity. These experiments were conducted in order to simulate under accelerated conditions the possible deterioration of the hematite pigment and plaster. The mineralogical transformation of the polychromy and plaster was monitored using mainly Raman spectroscopy, a non-destructive technique, but also infrared spectroscopy (FT-IR) and scanning electron microscopy energy-dispersive X-ray spectroscopy (SEM-EDS). After different exposure cycles to SO2, it was confirmed that hematite red pigment (Fe2O3) can be reduced into magnetite (Fe3O4), which provides the darkened colour to the pigment. While Fe(iii) from hematite is reduced into Fe(ii) or mixed Fe(iii) and Fe(ii), the SO2 can be oxidized (SO3) and hydrated to experience a subsequent wet deposition (H2SO4 aerosol) causing also the transformation of calcite into gypsum. Finally, it was assessed that high concentrations of SO2 can also cause the sulphation of hematite pigment promoting its transformation into paracoquimbite/coquimbite (Fe2(SO4)3·9H2O). Moreover, in some areas of the deteriorated painting fragments, non-expected iron(ii) sulphate and sulphite species were also identified.

Determination of the adulterants in adulterant–brandy blends using fluorescence spectroscopy and multivariate methods by Diana Markechová; Pavel Májek; Angela Kleinová; Jana Sádecká (379-386).
The addition of water or ethanol to brandy is an easy way to adulterate brandy. To avoid the misleading of consumers, it is necessary to develop a reliable method to detect the adulteration of brandy. In this work excitation–emission matrix fluorescence in combination with parallel factor analysis (PARAFAC) and partial least squares (PLS) regression was used to determine the content of water, ethanol and methanol in adulterated brandy samples. Excitation–emission matrix fluorescence spectra were measured in the emission wavelength range of 510–600 nm and in the excitation wavelength range of 393–497 nm. The model created using PARAFAC-PLS was able to predict the water, ethanol and methanol level in adulterated brandy with root mean square error of prediction values of 0.24%, 0.20% and 0.22%,respectively, and coefficients of determination of prediction between the reference content and the predicted values of 0.993, 0.997 and 0.995, respectively.

A combined spectroscopic study on Chinese porcelain containing ruan-cai colours by J. Van Pevenage; D. Lauwers; D. Herremans; E. Verhaeven; B. Vekemans; W. De Clercq; L. Vincze; L. Moens; P. Vandenabeele (387-394).
Because of its major contributions to the world economy and cultural heritage, Chinese porcelain and its technology can be considered as very important. In this research, the applicability of Raman spectroscopy and X-Ray Fluorescence (XRF) spectroscopy to study the pigments of the ruan-cai colour palette of Chinese porcelain dating from the 18th century was evaluated. Next to the investigations based on stylistic analysis, the use of other analysis techniques is important to obtain complementary information. Raman spectroscopy and XRF spectroscopy are two non-invasive, analytical techniques proven to be useful for the determination of the nature and composition of pigments used to decorate ceramics. As a result, analytical analysis allows us to retrieve more information about the production of Chinese porcelain. Based on visual inspection of the decoration, the objects could be divided into two groups and to evaluate the use of the combined analytical approach, all groups and subgroups were sampled. Before Raman and XRF analyses, the samples were cut, embedded and polished, in order to be able to measure the transversal section of the samples. In this way, each layer of the porcelain samples could be analysed separately. The results show that the blue colour is cobalt-aluminium oxide, in red zones haematite (α-Fe2O3) is present and the green area contains a mixture of CuO in a lead-rich basis and a solid solution of malachite (Cu2CO3(OH)2). The opaque yellow and white colours were identified as lead tin yellow type II (PbSn1−xSixO3, where x∼ 0.25) and a lead arsenate, respectively.

This study aimed at evaluating real-time, low-cost detection and quantification of bacteria, exemplified by E. coli O157 and Salmonella enterica serovar Typhimurium in aqueous systems. The approach takes advantage of the well-established latex-based immunoassay (LBI) and combines it with bacteria trapping and measuring using ultrasound. The results obtained demonstrate the utility of combining ultrasound standing waves of mean energy density 10 W cm−2, acoustic amplitude 100 kPa, and a laminar flow rate of the medium of 60 μl min−1 to trap selectively E. coli O157 or Salmonella enterica serovar Typhimurium (S. typhimurium) bacteria attached to 300–400 nm-antibody-coated latex particles. The ultrasound velocity measurements enabled a sensitivity down to 1.6 × 104 colony-forming units (CFU) ml−1 for E. coli O157 and 4.0 × 104 CFU ml−1 for S. typhimurium, compared to a sensitivity of 8.36 × 105 CFU ml−1 for E. coli O157 and 6.4 × 106 CFU ml−1 for S. typhimurium obtained by the standard LBI. The assay sensitivity can be further improved by use of various fluorescence methodologies.

Application of flower-like SnS2 nanoparticles for direct electrochemistry of hemoglobin and its electrocatalysis by Yan Zeng; Wei Li; Huanhuan Zhang; Xu Wu; Wei Sun; Zhihong Zhu; Ying Yu (404-409).
Flower-like SnS2 nanoparticles were synthesized through a hydrothermal process . By combing with hemoglobin (Hb) and the ionic liquid (IL) 1-butyl-3-methyl-imidazolium tetrafluoroborate ([BMIM]BF4), a composite biomaterial was fabricated and further used to modify a carbon ionic liquid electrode (CILE) with Nafion as a film-forming material. FT-IR and UV-Vis spectroscopic results showed that Hb retained its native structure in the composite. With the synergistic effects of the SnS2 nanoflower and IL, a couple of well-defined redox peaks of Hb appeared in the cyclic voltammogram of Nafion/Hb–SnS2–IL/CILE, which indicated that direct electron transfer between Hb and the underlying electrode was realized. The electrochemical behavior of Hb on the modified electrode was investigated by cyclic voltammetry with the electrochemical parameters such as electron transfer coefficient (α), electron transfer number (n) and heterogeneous electron transfer rate constant (ks) calculated. The fabricated electrode showed good electrocatalytic ability for the reduction of trichloroacetic acid (TCA) with a wider linear range from 0.8 to 21.0 mM and a lower detection limit of 0.27 mM (S/N = 3), indicating its potential application for the construction of a novel third-generation electrochemical biosensor.

A simple and new method for the simultaneous detection and quantification of vitamin C (ascorbic acid), phenolic acids (gallic acid and ellagic acid), hydroxycinnamic acid (chlorogenic acid) and flavonoids (myricetin, quercetin and kaempferol) in Emblica juice is developed. The compounds are separated in 18 minutes by an intangible curved gradient of 0.1% ortho-phosphoric acid in water (v/v) and acetonitrile, as mobile phase A and B, respectively, using Zorbax SB RP C-18 column at a wavelength of 254 nm. The assay was optimized by varying the mobile phase, gradient type, pretreatment method (thermal and non-thermal) and detection wavelength. The method was validated in terms of linearity, precision, detection limits and quantification limits. Good linear response was observed over the range specified for all the analytes, as confirmed by the correlation coefficient which ranged from 0.991 and 0.995. The limit of detection (LOD) and limit of quantification (LOQ) were found to be in the range of 0.129–0.685 μg ml−1 and 0.43–2.883 μg ml−1 respectively. Pulsed electric field (PEF) was used as the non-thermal pretreatment sample technique. HPLC-PDA showed increased levels of phenolic acids, as gallic and ellagic acid, and flavonoid, as quercetin, in PEF treated Emblica juice with respect to the untreated and thermally treated juice samples (p < 0.05). The overall precision values obtained for standards and samples were within the range 0.01–0.2 and 0.15–0.23 respectively. Further, total polyphenolic content and free radical scavenging capacity of the untreated, thermally and PEF treated Emblica juice were corroborated by HPLC.

In this study, two methods have been applied to the extraction of parabens from aqueous solution these are ionic liquid based aqueous two-phase extraction (IL-ATPS) and ionic liquid based aqueous two-phase extraction in the presence of β-cyclodextrin (β-CD) as the modifier (IL-βCD-ATPS). The IL-βCD-ATPS method consists of 1-butyl-3-methylimidazolium chloride, [C4mim][Cl] and β-CD. It is a new, green and simple extraction technique coupled with reversed-phase high performance liquid chromatography (RP-HPLC) that was applied to optimize conditions for the phase separation of parabens in water samples. The IL-βCD-ATPS method shows higher losses for water content compared to the IL-ATPS method resulting in a higher preconcentration factor and a higher distribution coefficient. A lower limit of detection of parabens in water samples was demonstrated by the IL-βCD-ATPS method. To conclude, the IL-βCD-ATPS method is superior to the IL-ATPS method for the determination of parabens in water samples. It is an economically viable approach when using β-CD because it improves the performance of the ATPS method dramatically. In addition, this chemical is cheap and non-toxic to the environment.

A novel method for the determination of trace formaldehyde in food samples has been established using a catalytic kinetic fluorimetry technique. It is based on the fact that the oxidation of pyronine Y by sodium periodate is catalyzed by formaldehyde in phosphoric acid. When the excitation wavelength was set at 546 nm, it was observed that decreased intensity (ΔF) of fluorescence at 563 nm was proportional to the concentration of formaldehyde in the range 0 to 0.30 μg mL−1. The limit of detection and the average recovery for formaldehyde were 2.0 × 10−2 ng mL−1 and 101.3% (n = 6), respectively. The present method has been applied to the determination of trace formaldehyde in aqueous products and the results observed were in good agreement with those obtained by distillation-spectrophotometry. A review of recently published catalytic or inhibiting kinetic methods for the determination of HCHO is presented for comparison. The mechanism of reaction is studied.

Validated LC-MS/MS method for simultaneous determination of Dasatinib and Sitagliptin in rat plasma and its application to pharmacokinetic study by Prinesh N. Patel; Gananadhamu Samanthula; Veeraraghavan Sridhar; Rambabu Arla; Kanthi Kiran V. S. Varanasi; Swaroop Kumar V.V.S (433-439).
Dasatinib (DST), a tyrosine kinase inhibitor, is a novel anticancer agent and Sitagliptin (STG) is an antidiabetic agent of dipeptidyl peptidase-IV inhibitor class. A novel, sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) based method has been developed for simultaneous monitoring of plasma levels of STG and DST in rat plasma. Both analytes and an internal standard (tolbutamide) were chromatographed on YMC-Pack ODS-AM (50 mm × 4.6 mm i.d., 3 μm) using a methanol : 2 mM ammonium acetate binary gradient mobile phase at a flow rate of 1 ml min−1 with a splitter (1 : 1) over a 5 min run time. Detection of analytes was performed on a LC-MS/MS system in multiple reaction monitoring (MRM) mode. The transitions monitored were 488.1 → 401.0, 408.1 → 174.0 and 271.1 → 155.0 for DST, STG and IS, respectively. The method was validated over a concentration range of 5.41–1029.60 ng ml−1 for DST and 5.64–1073.56 ng ml−1 for STG. The lower limit of quantification was 5.41 ng ml−1 and 5.64 ng ml−1 for DST and STG, respectively. Recoveries from spiked controls were >82% for the analytes and the internal standard at all QC levels. The intra- and inter-batch precision and accuracy across four quality control levels met established criteria of US Food and Drug Administration guidelines. This method was successfully applied to monitor the pharmacokinetic profile of both STG and DST in Wistar rats. This method can be applicable for pharmacokinetic drug–drug interaction studies.

Affinity molecules offer promise in the development of inexpensive, high-throughput methods that are complementary to traditional carbohydrate analysis techniques such as chromatography and mass spectrometry. Lectins are carbohydrate binding proteins which have been effectively used in many applications, however, their broad and label-dependent specificities can make data interpretation with known structures difficult and their unambiguous use for analysis of unknowns impossible. To evaluate the usefulness of lectins in distinguishing closely related structural glycoforms of glycoproteins, the well-characterised glycoprotein bovine fetuin (Fet), along with three additional Fet glycoform populations produced by enzymatic and chemical means, were profiled using a microarray consisting of 43 lectins with affinities covering a variety of carbohydrate structures. Fully-sialylated and fully-desialylated forms of Fet, as well as two intermediate forms with partial sialylation and galactosylation, generated distinct profiles. Using stringent data filtering, the total number of printed lectins required to distinguish four heterogeneous Fet glycoform pools was reduced to just eight and also enabled stronger correlation between known Fet glycan structures and reported lectin specificities. A major application of a high-throughput lectin profiling approach would be monitoring glycosylation on biopharmaceutical proteins, but a potential complication may be the presence of interfering molecules in the solvent matrix. This possibility was evaluated with an expanded dataset including an additional five partially-inhibitory conditions and the samples could also be individually discriminated with seven lectins using this strategy. Based on known carbohydrate structures, several lectins gave unexpected responses in the lectin microarray format. Two of the Fet glycoforms were also printed in a microarray format and profiled by a panel of thirteen fluorescently-labeled lectins to evaluate the performance and specificity of binding in another orientation. Seven lectins differed in behaviour between platforms which demonstrated that lectin performance is also format-dependent. Together, these findings demonstrate the utility of lectin microarray profiling for selective identification of glycoprotein glycoforms even with interfering molecules present. Also highlighted is the use of stringent data filtering to more accurately correlate profile data to glycan structure, as well as the importance of evaluating lectin performance and structural correlation in the intended platform.

An immunogold chromatographic assay was developed for quantitative determination of human chorionic gonadotropin (HCG) antigen. The monoclonal antibody to beta-HCG antigen (Mab II) conjugated gold nanoparticles (GNPs) were sprayed onto a conjugation pad for specific binding with the target protein to form an immunocomplex. The monoclonal antibody to alfa-HCG antigen (Mab I) was immobilized on the test line (T zone) of the nitrocellulose membrane (NC membrane) to capture the immunocomplex of gold nanoparticle labeled Mab II and HCG protein. Therefore, GNPs would aggregate on the test line of the NC membrane in the presence of HCG, which could be easily distinguished by the naked-eye. As for quantitative detection, the gray value of the red color in the T zone was proportional to the corresponding sample concentration. The gray value versus logarithm concentration curve presented a good linear relationship in the range of 10–600 ng mL−1. The duration of the assay was within 15 min and no professional large-scale analytical instrument was necessary for quantification. When applied in human serum analysis, the strips could reach the requirements of the clinic tests.

Determination of cocaine, its metabolites and pyrolytic products by LC-MS using a chemometric approach by Andrea Garcia Pereira; Felipe Bianchini D'Avila; Pâmela C. Lukasewicz Ferreira; Marcelo Gatteli Holler; Renata Pereira Limberger; Pedro Eduardo Fröehlich (456-462).
A method to assay cocaine (COC), its metabolites benzoylecgonine (BZE), ecgonine (ECG), ecgonine methyl esther (EME), and benzoylnorecgonine (BNE), pyrolytic products anhydroecgonine (AEC) and anhydroecgonine methyl ester (AEME) and adulterant levamisole (LEV) was developed and validated by liquid chromatography-mass spectrometry (LC-MS) using a chemometric approach including a two-level factorial design in the screening step and face-centered central composite design (FCCCD) to achieve the optimization. The method was carried out on positive electrospray ionization (ESI+) with a flow of 1 mL min−1 in isocratic mode consisting of 53% methanol and 47% ammonium acetate 10 mmol L−1 pH 6.3. The chromatographic separation was obtained with a Phenomenex Luna C18(2) column (250 mm × 4.6 mm, particle size 5 μm), with the temperature set at 31 °C. Validation parameters such as specificity, linearity, precision and accuracy were evaluated. The method was linear over the concentration range of 1–100 ng mL−1 for COC, AEME, EME, LEV, BZE and ECG and 5–100 ng mL−1 for AEC and BNE. The method was successfully applied to identify and quantify the analytes.

An aqueous two-phase extraction process composed of tetrahydrofuran/ammonium sulfate to extract nitrite from urine samples is investigated. The method is based on the reaction of nitrite with o-phenylenediamine in acid media to form benzotriazole and the aqueous two-phase extraction of benzotriazole in the presence of salt as an inducing agent in phase separation, followed by spectrofluorimetric detection. The system of fluorescence intensity was enhanced by complexation of hydroxypropyl-β-cyclodextrin. The optimal reaction and extraction conditions were studied. The linear calibration range for nitrite was 5–1000 ng mL−1 with a detection limit of 1.5 ng mL−1 (S/N = 3). The relative standard deviations for six replicate measurements varied between 0.3% and 1.7%. The interference effects of some anions and cations were also tested. The proposed method was successfully applied for the determination of nitrite in the urine samples with recoveries of 93.3% to 105.0%.

Anionic and cationic copolymerized ionic liquid-grafted silica as a multifunctional stationary phase for reversed-phase chromatography by Hongdeng Qiu; Mingliang Zhang; Jia Chen; Tongnian Gu; Makoto Takafuji; Hirotaka Ihara (469-475).
Recently, we briefly reported a new method to prepare ionic liquid-modified silica via copolymerization of anionic and cationic monomer couples on mercaptyl-modified silica, which aimed to enhance the stability of the column during the use of buffer as a solvent. In this paper, the previously prepared copolymerized ionic liquid-grafted silica denoted Sil-P(ImC18-SS) was characterized via elemental analysis, thermogravimetric analysis, and diffuse reflectance infrared Fourier transform spectroscopy. The reversed-phase chromatographic properties were determined particularly by separation of Tanaka test mixture, polycyclic aromatic hydrocarbons and their isomers, disubstituted benzenes, flavonoids, bases and nucleosides. Compared with the conventional ODS column, Sil-P(ImC18-SS) exhibited enhanced shape selectivity for polycyclic aromatic hydrocarbons and better performance for the separation of some polar analytes such as bases and flavonoids. Moreover, compared with previously reported ionic liquid-modified silica stationary phases, the anionic and cationic copolymerized stationary phase offers the advantage of increased stability given that the anions and cations of the ionic liquids were concomitantly immobilized on the silica.

The batch-to-batch quality consistency needs to be controlled to ensure the efficacy and safety of botanical drug products. Chromatographic fingerprinting is a powerful tool for characterizing complex chemical systems such as botanical drugs. This paper presented the combined use of a proposed weighting algorithm and multivariate statistical analysis on fingerprint data to achieve quality consistency evaluation of botanical drug products. A set of fingerprint data following multivariate normal distribution was simulated based on the real fingerprint data of a botanical drug product, Danshen injection. The weight of each peak was set based on its batch-to-batch peak variation. A principal component analysis (PCA) model was established, and the Hotelling T2 and DModX statistics were applied to evaluate quality consistency. The effect of weighting on the monitoring capability of the two statistics was evaluated through monitoring simulated peak area variations. The results showed that both the two statistics were more sensitive to the characteristic peaks with smaller batch-to-batch peak variations. Hence the peaks with larger variability will be permitted to have a wider tolerance range of variation, while a more narrow range of variation was seen for the peaks with smaller variability among batches. Peak weighting combined with multivariate statistical analysis has overcome some drawbacks of the fingerprint similarity analysis method, and is worthy of application and recommendation for the quality consistency evaluation of botanical drug products.

Double shell CdTe/CdS/ZnS quantum dots as a fluorescence probe for quetiapine determination in fumarate quetiapine tablets by Fangdi Wei; Yan Lin; Yanzi Wu; Xuan Sun; Liping Liu; Ping Zhou; Qin Hu (482-489).
A simple, sensitive and convenient method based on the fluorescence quenching of double shell CdTe/CdS/ZnS quantum dots (QDs) by quetiapine was proposed. CdTe/CdS/ZnS QDs were synthesized by a one-pot capping method in aqueous solution and characterized by absorption spectroscopy, fluorescence spectroscopy, X-ray photoelectron spectroscopy and transmission electron microscopy. Under optimal conditions, the calibration plot of F0/F versus the concentration of quetiapine was linear in the range of 5–100 μM with a correlation coefficient of 0.9958. The limit of detection was 0.8 μM. The concentration of quetiapine in quetiapine fumarate tablets was determined by the proposed method and the results agreed with the claimed values. The possible fluorescence quenching mechanism was discussed.

Glyphosate (GLYPH) is one of the most used herbicides around the world. In soil and waters, GLYPH is metabolized into aminomethylphosphonic acid (AMPA). Because of their high water solubility and half-life, both GLYPH and AMPA have been detected long after application. The present paper describes the development of a low cost Sequential Injection Chromatography (SIC) method for determination of GLYPH and AMPA based on pre-column oxidation of GLYPH to glycine (GLY) by hypochlorite, exploiting the stopped flow approach, followed by reaction with the fluorogenic reagent o-phthaldialdehyde in the presence of 2-mercaptoethanol (OPA-2ME) in borate buffer (pH > 9) to produce the fluorescent 1-(2′-hydroxyethylthio)-2-N-alkylisoindole. While GLYPH is being converted into GLY, the determination of AMPA is made by pre-column derivatization with OPA-2ME. Isocratic elution of both GLYPH and AMPA derivatives was done using a mobile phase composed of 20 : 80 (v/v) methanol : 10 mM phosphate buffer (pH 6.8) at a flow rate of 30 μL s−1. The proposed method solved the mutual fluorescence interference of AMPA and GLY derivatives, and the interference of humic substances. A linear response was observed for concentrations up to 5.0 μM for both compounds, with limits of detection and quantification of 0.11 and 0.35 μM (GLYPH), and 0.06 and 0.20 μM (AMPA), respectively. Application of the method to spiked natural waters gave recoveries between 78 and 134%, suggesting that the method has potential to be applied for water sampling trials.

A novel amperometric immunosensor for the detection of penicillin G (PeG) in milk was developed by covalent immobilization of new methylene blue (NMB) and Horseradish peroxidase-labeled penicillin polyclonal antibody (HRP-PePAb) on a glassy carbon electrode (GCE). The results of cyclic voltammetry and impedance spectroscopy showed that the NMB on the GCE exhibited excellent electrochemical activity and enhanced electron transfer. HRP labeled on PePAb shows remarkable electrocatalytic activity and good reproducibility towards the reduction of hydrogen peroxide due to the advantages of high electron transfer kinetics. Based on the specificity of the immunoreaction of antigen and antibody, the presented immunosensor exhibits high sensitivity, long-term stability, wide linear range, and low detection limit for the detection of PeG in milk. The linear response to PeG in the range of 5.20–41.6 nmol L−1 was obtained with the detection limit of 1.82 nmol L−1. Therefore, the proposed immunosensor can well be used to determine PeG residues in milk.

A GC/MS method for the simultaneous determination and quantification of chlorpromazine and diazepam in pork samples by Liqun Zhang; Pinggu Wu; Yiming Zhang; Quan Jin; Dajin Yang; Liyuan Wang; Jing Zhang (503-508).
A gas chromatography coupled with mass spectrometry (GC/MS) method for the simultaneous determination of chlorpromazine and diazepam in pork samples, which are two of the most often used antipsychotics, has been developed and validated. The samples were prepared and purified by solid-phase extraction (SPE), and then were analyzed by GC/MS. Chromatography was performed using an HP-5 MS 5% phenyl–95% methylpolysiloxane fused silica capillary column (30 m × 0.25 mm; film thickness 0.25 μm). The method had a linear calibration curve over the range 10–500 ng mL−1 (r > 0.99), recovery (>66.5% for chlorpromazine and >77.4% for diazepam), precision (%RSD < 11.5% for chlorpromazine and <9.5% for diazepam). The limit of quantification for both chlorpromazine and diazepam was 2.0 μg kg−1. This method can result in faster analysis, lower solvent consumption and less workload per sample while maintaining or even improving sensitivity.

Ambroxol hydrochloride (AMB), guaifenesin (GUF) and theophylline (THP) are co-formulated together for treatment of common cold and influenza. A newly developed spectrophotometric method; namely; ratio isoabsorptive point and ratio difference in subtracted spectra (RIDSS) spectrophotometric method was applied for resolving this ternary mixture with overlapping spectra in two steps. The proposed method is based on dividing the ternary mixture of the studied drugs by the spectrum of AMB to get the division spectrum, from which concentration of AMB can be obtained by measuring the amplitude values in the plateau region at 310 nm. Then the amplitude value of the plateau region at 310 nm was subtracted from the division spectrum and THP concentration was obtained by measuring the difference in amplitude values at 235 and 268 nm (corresponding to zero difference of GUF) while the total concentration of THP and GUF in the mixture was measured at their isoabsorptive point in the division spectrum at 235 nm (Aiso). GUF concentration is then obtained by subtraction. Validity of the method was tested by application to laboratory prepared mixtures and pharmaceutical formulations containing the studied drugs. Statistical comparison between the results of the proposed method and those of the reported HPLC method showed no significant difference.

Detection of pesticides in seized illegal cannabis plants by Serge Schneider; Roger Bebing; Carole Dauberschmidt (515-520).
The aim of this study was to screen seized illegal cannabis plants for pesticides. Cannabis samples were analysed according to the EN 15662 method. Pesticides were detected with UPLC/MS-MS in positive ESI mode using multiple reaction monitoring and GC-MS using scan mode. A total of 50 samples were tested for 160 different pesticides. Seven different pesticides were detected in 19 samples and five samples contained two pesticides. Four of the pesticides detected were fungicides (propamocarb, tebuconazole, propiconazole and tolylfluanid). The other 3 pesticides detected were insecticides or acaricides (imidacloprid, bifenthrin, and hexythiazox).

On the applicability of SiO2/Al2O3/Nb2O5 and SiO2/Al2O3/TiO2 as a biocompatible platform for chloroperoxidase by Márcia Simões Ribeiro; Fábio Jorge de Vasconcellos Júnior; Bruna Teixeira da Fonseca; Flávia Carvalho de Souza; Felipo Doval Rojas Soares; Éder Cláudio Lima; Murilo Feitosa Cabral; Emerson Schwingel Ribeiro; Eliane D'Elia (521-528).
In the present work, two mixed oxides, namely, SiO2/Al2O3/Nb2O5 and SiO2/Al2O3/TiO2 (designated as SiAlNb and SiAlTi, respectively), obtained using the sol–gel method were used to immobilize chloroperoxidase. Hydrogen peroxide was quantified using potassium hexacyanoferrate(ii) as a redox-mediator and amperometric measurements at 0.0 V vs. Ag/AgCl/Cl (3 M). The SiAlTi biosensor presented higher sensitivity than the SiAlNb biosensor, however, the first one did not present a good response regarding time. The developed biosensor using the SiAlNb mixed oxide provided good signal levels, good linearity, good stability (retaining approximately 70% of its original response after 6 weeks of usage), a low detection limit (3 μM), good sensitivity, a suitable working range (from 4 to 19 μM), fast response and good repeatability. The recovery of the amperometric method for the detection of hydrogen peroxide in synthetic samples was approximately 100 ± 2%, and for Listerine® Whitening Pre-Brush Rinse samples fortified with 1, 2 and 3% (v/v) of hydrogen peroxide, it was 100 ± 3%.

A new method was developed to detect fish freshness nondestructively by combining electronic nose (E-nose) and electronic tongue (E-tongue) in conjunction with chemometric methods. An E-nose with nine metal oxide semiconductor gas sensors and a commercial E-tongue were employed in this research. Pseudosciaena crocea stored at 4 °C for different days were used as experimental samples. Total viable counts (TVC) of the fish were detected by the conventional method. E-nose and E-tongue data were analyzed by principal component analysis. Three-layer radial basis function neural network (RBF-NN) models were established for qualitative discrimination of the fish freshness. Performances of RBF-NN models with different numbers of principal components (PCs) as the input were compared. Experimental results revealed that the best RBF-NN model was acquired at seven PCs of E-nose data with an optimal performance of 87.9% and 80.0% in the training set and prediction set respectively. While, the best RBF-NN model of E-tongue data analysis was at five PCs with an optimal performance of 86.3% in the training set and 81.8% in prediction set. Another RBF-NN model was built with the combination of E-nose and E-tongue. The result shows that the discrimination rates improved to 94.0% and 93.9% in the training set and prediction set respectively. A support vector machine regression model was applied to establish a relationship between the combined data from E-nose and E-tongue and from TVC values for quantitative determination. A high correlation was found between the merged data and the parameter of TVC with correlation coefficients more than 0.91. The results proved that, a single system of E-nose and E-tongue was enough to classify samples stored on different days at 4 °C, while a higher discrimination rate was acquired by the combination of the two sensors. The combined system could also be used to quantitatively evaluate the fish freshness. In conclusion, the combined system of E-nose and E-tongue in conjunction with appropriate chemometric analysis methods can conveniently and nondestructively evaluate the freshness of fish stored at 4 °C.

A novel high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method is developed for multiresidue analysis of twenty five synthetic colorants in meat matrices with matrix solid phase dispersion (MSPD). Good linearity was obtained with a correlation coefficient of 0.9935–0.9999. The quantitation limits (LOQs) for 12 acidic colorants were in the range from 0.89 μg kg−1 (acid orange 2) to 50.4 μg kg−1 (ponceau 4R) except for tartrazine, and the LOQs for 13 basic colorants were in the range from 0.52 μg kg−1 (Rhodamine B) to 4.19 μg kg−1 (disperse blue 7); among them the LOQ for 4 triphenylmethane colorants was in the range of 0.77–0.96 μg kg−1 which is better than the minimum required performance limit (MRPL) of 2 μg kg−1 for the sum of malachite green and leucomalachite green. The average recoveries for all analytes at three spiked levels were in the range of 75.4–123% except for Rhodamine B, (120–130%), with a RSD of <11%. The results showed that the content of 8 allowable food colorants and 17 banned synthetic colorants was lower than their LOQ level. This MSPD-HPLC-MS/MS method has the advantages of being rapid, sensitive, accurate, and high-throughput, and can be applied for multiresidue analysis of synthetic colorants in meat products.

Generation of single-stranded DNA plays a key role in many biotechnology applications including production of aptamers, single strand conformation polymorphism, nuclease S1 mapping, pyrosequencing, genosensors, probe preparation and labelling, subtractive hybridization as well as nucleic acid sensing and microarrays. Several methods are available in the literature to produce single-stranded DNA from double-stranded DNA templates, such as extraction of the sense strand from denaturing gels, asymmetric PCR, use of streptavidin–biotin interaction, and some alternative methods, including enzymatic digestion of the negative strand by either lambda exonuclease or T7 Gene 6 exonuclease. In this report, a detailed characterization of a dual biotin tag method to generate single-stranded DNA from the random oligonucleotide library is presented. Unlike the traditional streptavidin–biotin method that uses single biotin tagged molecules during separation, this novel technique is based on a dual biotin molecule covalently attached to the 5′ end of the negative strand. The improved technique takes less than one hour as a consequence of the eliminated alkali treatment step, which makes this procedure the shortest procedure described in the literature so far for single-stranded DNA production. The method can achieve a single-stranded DNA yield around 75% from the corresponding DNA template in Tris–HCl buffer. A number of parameters, such as the effect of different elution buffers and heat treatments, spontaneous release of streptavidin from the magnetic bead surface, loss of beads during consecutive washes, aggregation of the beads, were investigated to reveal the optimal conditions for single-stranded DNA production. FTIR, DLS, SEM, and electrophoresis techniques were used for characterization studies.

Protein identification by mass spectrometry forms the cornerstone of proteomics. Commonly, the identification of proteins is based on digestion of proteins into peptides using proteolytic enzymes. Cleavage of the disulphide bonds in proteins before enzymatic digestion and mass spectral analysis is important in order to facilitate the accessibility of the enzyme to cleave the proteins into peptides. As a result, the protein sequence coverage will be increased. In this study, a novel approach for immobilisation of the reducing reagent on the surface of a silica-based monolith in order to use it for reduction of disulphide bonds in proteins was successfully developed. This was carried out by silanisation of the surface of the silica-based monolith with (3-aminopropyl)triethoxysilane (APTES), followed by immobilisation of the reducing reagent, tris(2-carboxyethyl)phosphine hydrochloride (TCEP) on the surface of the amino-bonded silica monolith. The fabricated monolith was characterised using SEM analysis, EDX analysis, IR spectroscopy, and BET model. The performance of the fabricated glass microchip containing the TCEP-immobilised silica monolith to reduce the disulphide bonds in proteins was checked by injection of 100 μL of denatured insulin inside the microchip using a syringe pump at a flow rate of 10 μL min−1, followed by sealing both ends of the ETFE tubes with Blu-Tak. The microchip was kept in a humidified chamber for 30 min at 60 °C. After the reduction reaction, the reduced cysteine residues were alkylated with IAA at 60 °C for 30 min, followed by using a MALDI-TOF-MS instrument for qualitative confirmation. The results show that the fabricated microchip-based silica monolith has the ability to reduce disulphide bonds in insulin. In addition, the method is simple, reduces the risk of contamination, and results in lower amounts of the sample and reagents compared with the conventional techniques for proteomics sample preparation. A future study investigating reduction of the disulphide bonds in proteins from a real sample using this new microfluidic device would be very interesting.

Polydimethylsiloxane (PDMS) tube microextraction for the determination of 2,3-dimethylphenol (2,3-DMP), 2,6-di-tert-butylphenol (2,6-DTBP) and 4,4′-(1-methylethyliden)bisphenol (4,4′-(1-M)BP) in root exudates of hot pepper (Capsicum annuum L.) by gas chromatography-mass spectrometry (GC-MS) was studied by optimizing derivatization and extraction conditions. Optimum extraction parameters were obtained including PFPy as derivative reagent; derivative reagent volume of 200 μL; sample solution pH of 11; extraction time of 30 min; extraction temperature of 60 °C; isooctane as elution reagent; elution reagent of 1.5 mL. The developed method shows good linearity levels in the range of 0.2–20 μg mL−1 with correlation coefficients of 0.9972, 0.9985 and 0.9978, and relative standard deviations in the range of 4.8–8.4% for 2,3-DMP, 2,6-DTBP and 4,4′-(1-M)BP, respectively, and it's extraction performance was better than that of SPE method. Recoveries ranging from 85.7 to 103.9% and the RSD below 7.9% were obtained when optimized method was applied to determine 2,3-DMP, 2,6-DTBP and 4,4′-(1-M)BP for 15 and 30 days' root hydroponic solution of hot pepper. The developed method is rapid, simple and inexpensive and offers high sensitivity, good reproducibility and satisfactory recovery.

Fluorescence quenching by copper(ii) of a Schiff base derivative of p-tert-butylcalix[4]arene (SANCAX) in 0.1% polyethylene glycol tert-octylphenyl ether (Triton X-100) medium was investigated. The fluorescence of SANCAX is quenched by Cu2+ due to the formation of a weaker fluorescent inclusion complex (Cu-SANCAX). The fluorescence quenching (ΔF = FSANCAX−FCu-SANCAX) is sensitized in Triton X-100. Under optimal conditions, the linear range calibration curve for the determination of Cu2+ is 0.005–0.08 μg mL−1. The detection limit is 3.05 ng mL−1 with an RSD of 1.55% (n = 3, c = 0.05 μg mL−1). The SANCAX fluorescence quenching mechanism was investigated by the Benesi–Hildebrand method, fluorescence quantum yield, and IR. The method has been applied in the determination of trace Cu2+ in real samples with satisfactory results.

A UV-Vis spectrophotometric method for characterization of plant extracts with specific acid dissociation constants was developed and nine traditional Chinese plant extracts were used in the analysis workflow. The ionization of plant extracts gave intensive pH-dependent spectral shifts and their spectral changes differed between wavelength ranges. The simulation with the one-step and two-step ionization processes showed that the acid dissociation constants distributed steadily at some wavelength ranges. These specific acid dissociation constants might be used as markers for characterization of plant extracts. This study suggests that the proposed method has potential applications for quality assessment and mass quality control of plant extracts used in food and pharmaceutical industries.

A porous functional polymer was prepared by in situ polymerization reaction. In this process, azobisisobutyronitrile was used as the initiator; hexanediol diacrylate (HDDA) and butyl methacrylate (BMA) as co-monomers; dodecyl alcohol as the porogenic solvent. The conditions of polymerization were optimized. The morphology of the polymer was investigated by scanning electron microscopy. Chemical groups in the monolith were assayed by infrared spectrometry. The pore size distribution was determined by mercury intrusion porosimetry and nitrogen adsorption–desorption. Finally, the functional polymer, a poly(HDDA-co-BMA-co-EDMA) monolith, was used as the stationary phase of high performance liquid chromatography (HPLC) to separate small molecules.

Rapid chemical analysis of bear bile: 5 minute separation and quantitation of bile acids using UHPLC–qTOF-MS by Xue Qiao; Wei Song; Xiong-hao Lin; Qi Wang; Tao Bo; De-an Guo; Junxiu Liu; Min Ye (596-601).
Bear bile is a precious traditional Chinese medicine containing abundant bile acids. The content of these bile acids varies significantly. In this study, a rapid and reliable method was established to simultaneously monitor major and minor bile acids, using ultra-high performance liquid chromatography coupled with quadruple time-of-flight mass spectrometry. The samples were separated on a 1.8 μm Zorbax Eclipse-Plus C18 column with acetonitrile and water (containing 4 mM of ammonium acetate) as the mobile phase. A 5 min analysis allowed the characterization of 21 steroids and the quantitation of two major constituents, taurochenodeoxycholic acid (TCDCA) and tauroursodeoxycholic acid (TUDCA). The contents of TUDCA and TCDCA in 8 commercial batches of bear bile were 39.04 ± 7.83% and 36.10 ± 9.42% (w/w; mean ± standard deviation), respectively, and the ratio of TUDCA/TCDCA ranged from 0.82 to 1.76. The simultaneous qualitative and quantitative analyses of bear bile powder was achieved, and minor steroid constituents were identified.

A molecularly imprinted polymer (MIP) monolith was initially prepared in a silica capillary mold by microwave irradiation. After the silica wall was etched away after polymerization, the resulting monolithic bar was embedded in a porous hollow fiber membrane tube (HFMT), in which a thin supported liquid membrane (SLM) was formed in the pores and an acceptor solution was filled in its lumen. It can be used to overcome the common water-compatibility problem in the MIP application by the developed hollow fiber–liquid–solid microextraction (HFLSME) system. The main factors affecting the selective extraction including extraction and desorption time, extraction solvent in the hollow fiber (HF) lumen, off-line desorption solvent, stirring rate and pH of the sample solution were investigated for HFLSME procedures in detail. Selectivity was also evaluated using the atrazine template molecule, its analogues and non-analogue. The extraction amount for each structurally-related pesticide including atrazine, 2-amino-4-methoxy-6-methyl-1,3,5-triazine, terbutylazine and ametryn was obtained about 10 times using atrazine-imprinted hollow fiber than that using non-imprinted hollow fiber, whilst no obvious difference was found for its non-analogues of metribuzin. The resulted hollow fibers were successfully applied to detect atrazine and its analogues in lake water coupled to high performance liquid chromatography (HPLC) and resulted in recoveries in the range of 72.8–113.2%, respectively.

An efficient ratiometric fluorescent excimer probe for hypochlorite based on a cofacial xanthene-bridged bispyrene by Cui-Cui Zhang; Yijun Gong; Yuan Yuan; Aili Luo; Weijun Zhang; Jing Zhang; Xiaobing Zhang; Weihong Tan (609-614).
In this paper, by employing a rigid xanthene scaffold as a bridge, we for the first time report a cofacial bispyrene derivative DPH as an efficient ratiometric fluorescent excimer probe for hypochlorite (OCl). The probe is comprised of a rigid xanthene scaffold and two parallel pyrenes, which are linked by an OCl-sensitive dicarboxylic acid hydrazide group. The introduction of OCl, however, will induce oxidation of the dicarboxylic acid hydrazide moiety into a diimide group, and the subsequent hydrolysis of the diimide, to give 1-pyrenecarboxylic acid which exhibits a monomer emission at about 380 nm, with its intensity increasing with the addition of increased concentration of OCl. Meanwhile, the excimer emission intensity gradually decreases. Such a ratiometric fluorescent response of the probe affords a high sensitivity to OCl, with a linear response concentration range of 1 to 300 μM, and a detection limit of 0.35 μM for OCl. It also shows a high selectivity for OCl with no interference observed from other common anions and small molecules. Moreover, it can also act as a colorimetric probe for OCl due to the cyan-to-blue fluorescence color change. It has been preliminarily used for practical detection of OCl in river water samples with satisfying results.

Stability-indicating spectroflurometric method for determination of triclabendazole in pure form and tablets by Fathalla F. Belal; Mohie K. Sharaf El-Din; Nahed M. El Enany; Samar Saad (615-622).
A simple, rapid and highly sensitive spectrofluorimetric method was applied in the determination of triclabendazole (TCB). The method depends on measuring the native fluorescence of TCB in methanol at 338 nm after excitation at 298 nm. The method was rectilinear over the concentration range of 0.05–0.9 μg mL−1 with a limit of detection 13.2 ng mL−1 and limit of quantification of 39.9 ng mL−1. The method was fully validated and successfully applied for the determination of TCB in its tablets with an average percentage recovery of 98.73 ± 1.11. The method was further extended for the stability study of TCB. The drug was exposed to acidic, alkaline, oxidative and UV degradation according to International Conference on Harmonization guidelines. Moreover the method was utilized to investigate the kinetics of the acidic, photolytic and oxidative degradation of the drug. The reaction rate constants and half times were also calculated. A proposal for the degradation pathways was postulated.

In vitro cell models are easy, reproducible, and cost-effective tools for tracking drug absorption rates and for elucidating related mechanisms. Traditionally, before introduction into an analysis system, samples crossing an in vitro cell membrane usually undergo complicated processes including precipitation, centrifugation, and even filtering. In the current study, a generic, sensitive and rapid method was developed for the determination of natural products in the matrix of in vitro drug absorption systems (HBSS, Hank's balanced salt solution). A guard RP-C18 column was used to retain the target compounds, while a tandem mass spectrometry system in the multiple reaction monitoring mode was used to specifically detect the target compounds. In addition, a six-way valve was used to connect the LC and MS systems, and to automatically switch the flow directions between the mass spectrometer and the waste. The method was validated by determining the accuracy, precision and sensitivity using seven natural products including (+)-praeruptorin A (dPA), (−)-praeruptorin A (lPA), (+)-praeruptorin B (dPB), (−)-praeruptorin B (lPB) from Peucedani Radix (Chinese name: Qian-hu), morusin (Mo), sanggenon C (SC), and kuwanon G (KG) from Mori Cortex (Chinese name: Sang-Bai-Pi). The developed method is proposed to be applied for in vitro absorption and transport studies.

Back cover (629-630).