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Analytica Chimica Acta (v.711, #)

Editorial Board (pp. iii).

Raman spectroscopy for forensic examination of β-ketophenethylamine “legal highs”: Reference and seized samples of cathinone derivatives by Samantha P. Stewart; Steven E.J. Bell; Nicholas C. Fletcher; Samira Bouazzaoui; Yen Cheng Ho; S. James Speers; K. Laota Peters (pp. 1-6).

Display Omitted► We present Raman spectra of nine different β-ketophenethylamine reference standards. ► Substitution on the aromatic ring affects the Raman spectrum in a predictable way. ► The spectra can thus be used for rapid identification of this class of “legal highs”. ► Seized samples show bands from both the drug and bulking agents. ► This allows the composition of street quality samples to be determined.Raman spectra of a representative range of β-ketophenethylamine (β-KP), the rapidly growing family of cathinone-related “legal high” recreational drugs, have been recorded. These spectra showed characteristic changes that were associated with the pattern of substitution on the aromatic rings, for example, the compounds carrying substituents at the 4- position could be distinguished from 3,4-methylenedioxy “ecstasy” derivatives. They also showed small but detectable changes with differences in substitution on the ethylamine substituent. These features allowed the β-KPs present in seized casework samples to be identified. The seized samples typically contained only small amounts of bulking agents, which meant that the band intensities of these components within averaged data were very small. In contrast, grid sampling normally gave at least some spectra which had a higher than average proportion of the bulking agent(s), which allowed them to also be identified. This study therefore demonstrates that Raman spectroscopy can be used both to provide a rapid, non-destructive technique for identification of this class of drugs in seized samples and to detect minor constituents, giving a composition profile which can be used for drugs intelligence work.

Keywords: Raman; Phenethylamines; Beta-keto; Legal high; Ecstasy and cathinone

Liquid chromatography–mass spectrometry based global metabolite profiling: A review by Georgios A. Theodoridis; Helen G. Gika; Elizabeth J. Want; Ian D. Wilson (pp. 7-16).

Display Omitted► LC–MS is increasingly used in global metabolite profiling despite stability issues. ► Data extraction and marker identification remain at the centre of interest. ► Pre-analytical issues and sample preparation are also important factors. ► Method validation and protocol harmonisation are needed to obtain quality data. ► Large scale efforts and their outcome (e.g. HMDB) provide breakthrough paradigms.Untargeted, global metabolite profiling (often described as metabonomics or metabolomics) represents an expanding research topic and is, potentially, a major pillar for systems biology studies. To obtain holistic metabolic profiles from complex samples, such as biological fluids or tissue extracts, requires powerful, high resolution and information-rich analytical methods and for this spectroscopic technologies are generally used. Mass spectrometry, coupled to liquid chromatography (LC–MS), is increasingly being used for such investigations as a result of the significant advances in both technologies over the past decade. Here we try to critically review the topic of LC–MS-based global metabolic profiling and describe and compare the results offered by different analytical strategies and technologies. This review highlights the current challenges, limitations and opportunities of the current methodology.

Keywords: Metabolite profiling; LC–MS; Metabonomics; Metabolomics; Biomarker discovery

Nanogold-functionalized magnetic beads with redox activity for sensitive electrochemical immunoassay of thyroid-stimulating hormone by Bing Zhang; Dianping Tang; Bingqian Liu; Yuling Cui; Huafeng Chen; Guonan Chen (pp. 17-23).

Display Omitted► We designed an electrochemical immunoassay of thyroid-stimulating hormone with signal amplification. ► A new signal tag with biofunctionalized organic–inorganic GoldMag nanostructures. ► A well-dispersive gold nanoparticles on graphene nanosheets. ► Redox-active GoldMag nanolabels.A new electrochemical immunosensor for sensitive determination of thyroid-stimulating hormone (TSH) was designed by using redox-active nanogold-functionalized magnetic beads (GoldMag) as signal tags on the nanogold–graphene interface. To construct such GoldMag nanostructures, polyethyleneimine-functionalized magnetic beads (PEI-MBs) were initially prepared by using a wet chemical method, and the electroactive thionine molecules and gold nanoparticles were then alternately immobilized on the surface of PEI-MBs by using an opposite-charged adsorption technique and an in situ synthesis method, respectively. The synthesized GoldMag nanostructures were utilized as signal tags for the label of horseradish peroxidase-anti-TSH conjugates (HRP-anti-TSH). With a sandwich-type immunoassay format, the conjugated signal tags on the transducer were increased with the increasing TSH concentration in the sample, thus enhancing the signal of the electrochemical immunosensor due to the labeled HRP toward the catalytic reduction of H₂O₂. Under optimal conditions, the current was proportional to the logarithm of TSH concentration ranging from 0.01 to 20μIUmL⁻¹ in pH 6.0 HAc–NaAc containing 6mM H₂O₂. The detection limit (LOD) was 0.005μIUmL⁻¹ TSH at 3 s_B. The immunosensor displayed an acceptable reproducibility, stability and selectivity. In addition, the methodology was evaluated with human serum specimens, receiving good correlation with results from commercially available electrochemiluminescent analyzer.

Keywords: Electrochemical immunosensor; Gold–graphene nanocomposites; Nanolabels; Redox-active GoldMag nanostructures; Thyroid-stimulating hormone

Electrochemical bisphenol A sensor based on N-doped graphene sheets by Haixia Fan; Yan Li; Dan Wu; Hongmin Ma; Kexia Mao; Dawei Fan; Bin Du; He Li; Qin Wei (pp. 24-28).

Display Omitted► N-doped graphene sheets have catalytic activity towards the BPA oxidation. ► The biosensor based on N-doped graphene sheets and chitosan. ► This method was proposed for determination of BPA utilizing N-doped graphene sheets.Bisphenol A (BPA), which could disrupt endocrine system and cause cancer, has been considered as an endocrine disruptor. Therefore, it is very important and necessary to develop a sensitive and selective method for detection of BPA. Herein, nitrogen-doped graphene sheets (N-GS) and chitosan (CS) were used to prepare electrochemical BPA sensor. Compared with graphene, N-GS has favorable electron transfer ability and electrocatalytic property, which could enhance the response signal towards BPA. CS also exhibits excellent film forming ability and improves the electrochemical behavior of N-GS modified electrode. The sensor exhibits a sensitive response to BPA in the range of 1.0×10⁻⁸–1.3×10⁻⁶molL⁻¹ with a low detection limit of 5.0×10⁻⁹molL⁻¹ under the optimal conditions. Finally, this proposed sensor was successfully employed to determine BPA in water samples with satisfactory results.

Keywords: N-doped graphene; Electrochemical sensor; Bisphenol A; Endocrine disruptor

Number of graphene layers exhibiting an influence on oxidation of DNA bases: Analytical parameters by Madeline Shuhua Goh; Martin Pumera (pp. 29-31).

Display Omitted► Double-layer, few-layer and multilayer graphene sheets were used as electrochemical surfaces. ► Guanine and adenine electrooxidation was studied in the presence of cytosine and thymine. ► The number of graphene layers have profound influence on electrochemical oxidation of DNA bases. ► Few-layer graphene provides the most sensitive response.This article investigates the analytical performance of double-, few- and multi-layer graphene upon oxidation of adenine and guanine. We observed that the sensitivity of differential pulse voltammetric response of guanine and adenine is significantly higher at few-layer graphene surface than single-layer graphene. We use glassy carbon electrode as substrate coated with graphenes. Our findings shall have profound influence on construction of graphene based genosensors.

Keywords: Graphene; Electrochemistry; DNA bases; Graphite

Electrochemical detection of hydrazine using a highly sensitive nanoporous gold electrode by Ying-Yao Tang; Chai-Lin Kao; Po-Yu Chen (pp. 32-39).

A facile alloy–dealloy procedure has been developed to prepare nanoporous Au (NPG) electrode for highly sensitive electrochemical detection of hydrazine.Display Omitted► A facile alloy–dealloy procedure was developed to prepare nanoporous gold (NPG) electrode in aqueous solution. ► Highly sensitive detection of hydrazine was achieved using the NPG electrode. ► NPG electrode demonstrated an unusual surface-confined voltammetric behavior. ► The nanopores may behave like thin-layer cells, leading to the surface-confine manner.A facile alloy–dealloy technique performed in aqueous media was employed to prepare a nanoporous gold (NPG) electrode that demonstrated extremely high sensitivity toward hydrazine oxidation. An Ag_∼60Au_∼40 alloy was electrodeposited at a constant potential on sequentially Cr- and Au-deposited indium tin oxide (Au/Cr/ITO) from a bath that contained sulfuric acid, thiourea, HAuCl₄·3H₂O, and AgNO₃. The dealloying step was performed in concentrated HNO₃, where Ag in the alloy was selectively oxidized to leave the NPG structure. The NPG electrode was employed to study the hydrazine oxidation in basic phosphate buffer solution (PBS), and the results were compared with those obtained using the gold nanoparticle (AuNP)-modified ITO (AuNP/ITO) electrode. The NPG electrode demonstrated an unusual surface-confined behavior, which probably resulted from the thin-layer characteristics of the nano-pores. Hydrazine was detected by hydrodynamic chronoamperometry (HCA) at +0.2V (vs. Ag/AgCl). The steady-state oxidative current exhibited a linear dependence on the hydrazine concentration in the concentration range of 5.00nM–2.05mM, and the detection limit was 4.37nM ( σ=3). This detection limit is the lower than the detection limits reported in the current literature concerning the electrochemical detection of hydrazine. The NPG electrode indeed demonstrates greater stability after hydrazine detection than the AuNP/ITO electrode.

Keywords: Nanoporous; Electrodeposition; Alloy–dealloy; Hydrazine; Electrochemical detection

A simple and an efficient strategy to synthesize multi-component nanocomposites for biosensor applications by Xiaoquan Lu; Yan Li; Xia Zhang; Jie Du; Xibin Zhou; Zhonghua Xue; Xiuhui Liu (pp. 40-45).

This scheme is a brief synthetic procedure and the chemical structure of the Au–PPy/PB core-shell nanocomposites. With the addition of pyrrole monomers, pyrrole was autopolymerization to PPy and AuCl₄⁻ was reduced to elemental Au instantaneously and simultaneously, and more excitedly, PB was produced along with elemental Au serving as a catalyst.Display Omitted► Au–PPy/PB nanocomposites can be grown quickly and directly by a one-step method. ► There is no adding any reductant and stabilizer in the synthesis progress. ► The novel one-step method is facile, environmentally benign and available. ► Au–PPy/PB nanocomposites are used to construct a stable and sensitive H₂O₂ sensor.We demonstrate that core–shell multi-component nanocomposites can be grown in situ at room temperature by a novel one-step approach without adding any reductant and stabilizer. We have presented a one-step method for the synthesis of multi-component nanocomposites in water solution, the multi-component nanocomposites could be produced directly and quickly in an in situ wet-chemical reaction. Here, Au–polypyrrole (PPy)/Prussian blue (PB) nanocomposites have been synthesized successfully under the same circumstance. With the addition of pyrrole monomers into mixture solutions, the autopolymerization of pyrrole into PPy and AuCl₄⁻ was reduced to elemental Au instantaneously as well as simultaneously. At the same time, PB produced along with elemental Au serving as a catalyst. Furthermore, we investigated the performance of Au–PPy/PB nanocomposites as amperometric sensor toward the reduction of H₂O₂, which displayed high sensitivity, fast response and good stability. The peak current of H₂O₂ increased linearly with the concentration of H₂O₂ in the range from 2.5×10⁻⁹ to 1.2×10⁻⁶M, and the low detection limit of 8.3×10⁻¹⁰M (S/N=3) was obtained. Therefore, this work provides a new pathway to design and fabricate novel multi-component nanocomposites, which have unique characteristics and hold great applications in the fields of sensors, electrocatalysis and others.

Keywords: One-step; Au–polypyrrole (PPy)/Prussian blue (PB); Multi-component nanocomposites; Biosensor

Determination of aminoglycoside residues by liquid chromatography and tandem mass spectrometry in a variety of matrices by A. Kaufmann; P. Butcher; K. Maden (pp. 46-53).

Display Omitted► We are reporting a quantitative multi-residue method for aminoglycoside antibiotics. ► The use of a strong cation exchanger step significantly simplifies the clean-up. ► A novel eluent permits the quantitative elution of otherwise retained analytes. ► The proposed elution regime should be relevant for other strongly retained compounds.A quantitative LC–MS/MS method was developed for the determination of 13 commonly used aminoglycoside antibiotics in meat (pork muscle, fish, and veal livers and kidneys). The proposed method is sufficiently sensitive and highly selective. Unlike other previously reported methods, it uses a simple clean-up procedure based on a strong cation-exchange solid-phase cartridge that permits high sample extract loading volumes. A unique elution regime based on a volatile buffer at intermediately high pH value in combination with an organic solvent provides quantitative elution of the various aminoglycosides. This methodology ensured that neither a breakthrough of weakly retained aminoglycosides (e.g. spectinomycin) nor the incomplete elution of strongly retained analytes (e.g. neo- and gentamycin) is observed. The single-step clean-up is fast and produces clean extracts that minimize matrix-related signal suppression in the electrospray interface.

Keywords: Tandem mass spectrometry; Veterinary drugs; Aminoglycosides; Solid phase extraction

H, C, N and S stable isotopes and mineral profiles to objectively guarantee the authenticity of grated hard cheeses by Federica Camin; Ron Wehrens; Daniela Bertoldi; Luana Bontempo; Luca Ziller; Matteo Perini; Giorgio Nicolini; Marco Nocetti; Roberto Larcher (pp. 54-59).

. Random Forest model based on δ¹³C, δ²H, δ¹⁵N, δ³⁴S and the content of Sr, Cu, Mo, Re, Na, U, Bi, Ni, Fe, Mn, Ga, Se, Er, Dy, Pb, Li, usable for the protection of PDO Parmigiano Reggiano cheese from mislabelling. The correct classification rate in cross-validation is 98.6%.Display Omitted► The isotopic and elemental profile of over 260 hard cheese samples are discussed. ► Two validated and immediately applicable statistical models are presented. ► One model is able to predict the origin of seven types of European hard cheeses. ► The other one allows to discriminate the PDO Parmigiano Reggiano cheese from imitators. ► The most significant variables are δ¹³C, δ²H, δ¹⁵N, δ³⁴S and the content of 16 elements.In compliance with the European law (EC No. 510/2006), geographical indications and designations of origin for agricultural products and foodstuffs must be protected against mislabelling. This is particularly important for PDO hard cheeses, as Parmigiano Reggiano, that can cost up to the double of the no-PDO competitors.This paper presents two statistical models, based on isotopic and elemental composition, able to trace the origin of cheese also in grated and shredded forms, for which it is not possible to check the logo fire-marked on the rind. One model is able to predict the origin of seven types of European hard cheeses (in a validation step, 236 samples out of 240 are correctly recognised) and the other specifically to discriminate the PDO Parmigiano Reggiano cheese from 9 European and 2 extra-European imitators (260 out of 264 correct classifications). Both models are based on Random Forests. The most significant variables for cheese traceability common in both models are δ¹³C, δ²H, δ¹⁵N, δ³⁴S and Sr, Cu, Mo, Re, Na, U, Bi, Ni, Fe, Mn, Ga, Se, and Li. These variables are linked not only to geography, but also to cow diet and cheese making processes.

Keywords: IRMS; ICPMS; Traceability model; Parmigiano Reggiano; Mislabelling

Analysis of multiplex endogenous estrogen metabolites in human urine using ultra-fast liquid chromatography–tandem mass spectrometry: A case study for breast cancer by Jiang Huang; Jianghao Sun; Yanhua Chen; Yongmei Song; Lijia Dong; Qinmin Zhan; Ruiping Zhang; Zeper Abliz (pp. 60-68).

A rapid, sensitive, specific and accurate analytical method of ultra-fast liquid chromatography combined with tandem mass spectrometry (UFLC–MS/MS) was established for simultaneous quantitative analysis of 16 distinct endogenous estrogens and their metabolites (EMs) in postmenopausal female urine. The quantitative method utilized a hydrolysis/extraction/derivatization step and a UFLC system to achieve separation in 16min. The lower limit of quantitation for each estrogen metabolite was 2pgmL⁻¹ with the percent recovery of a known added amount of estrogen at 93.2–109.3%. The intra-batch accuracy and precision for all analytes were 87.5–107.7% and 0.6–11.7%, respectively, while inter-batch accuracy and precision were 87.0–105.8% and 1.2–10.2%, respectively. Using this developed and validated method, the comprehensive metabolic profiling of 16 EMs in urine samples of 86 postmenopausal female breast cancer patients and 36 healthy controls was investigated by systematic statistical analysis. As a result, the circulating levels of 6 EMs were found to be different by a comparison of patients and healthy controls. The parent estrogens, estrone (E1) and 17β-estradiol (E2), as well as 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) were produced in higher abundance, whereas 16α-hydroxyestrone (16α-OHE1) and 2-methoxyestradiol (2-MeOE2) were decreased in the breast cancer group. 2-OHE2 and 4-OHE2 in particular showed significant elevation in patients, which are consistent with the carcinogenic mechanism hypothesis that catechol estrogens can react with DNA via quinones, resulting in mutations to induce breast cancer. Thus, 2,4-hydroxylation may be the dominant metabolic pathway for parent estrogens rather than 16α-hydroxylation. The lower level of 2-MeOE2 in the breast cancer group was believed to correlate with its protective effect against tumor formation. This study could provide valuable information on the association of the EM metabolic pathway with carcinogenesis as well as identify potential biomarkers for estrogen-induced breast cancer risk.

Keywords: Estrogen metabolite; Breast cancer; Urine; Ultra-fast liquid chromatography–tandem mass spectrometry; Metabolic profiling

Microchip capillary electrophoresis–electrospray ionization–mass spectrometry of intact proteins using uncoated Ormocomp microchips by Tiina Sikanen; Susanna Aura; Sami Franssila; Tapio Kotiaho; Risto Kostiainen (pp. 69-76).

Display Omitted► We integrated Ormocomp-based microchip electrophoresis to mass spectrometry. ► Intact proteins were analyzed without any surface modification of the microchips. ► Ormocomp microchips enable efficient separation of both peptides and proteins.We present rapid (<5min) and efficient intact protein analysis by mass spectrometry (MS) using fully microfabricated and monolithically integrated capillary electrophoresis–electrospray ionization (CE–ESI) microchips. The microchips are fabricated fully of commercial inorganic–organic hybrid material, Ormocomp, by UV-embossing and adhesive Ormocomp–Ormocomp bonding (CE microchannels). A sheath-flow ESI interface is monolithically integrated with the UV-embossed separation channels by cutting a rectangular emitter tip in the end with a dicing saw. As a result, electrospray was produced from the corner of chip with good reproducibility between parallel tips (stability within 3.8–9.2% RSD). Thanks to its inherent biocompatibility and stable (negative) surface charge, Ormocomp microchips enable efficient intact protein analysis with up to ∼10⁴ theoretical separation plates per meter without any chemical or physical surface modification before analysis. The same microchip setup is also feasible for rapid peptide sequencing and mass fingerprinting and shows excellent migration time repeatability from run to run for both peptides (5.6–5.9% RSD, n=4) and intact proteins (1.3–7.5% RSD, n=3). Thus, the Ormocomp microchips provide a versatile new tool for MS-based proteomics. Particularly, the feasibility of the Ormocomp chips for rapid analysis of intact proteins with such a simple setup is a valuable increment to the current technology.

Keywords: Organically modified ceramics (ORMOCERs); Microfluidics; Microchip electrophoresis; Electrospray ionization; Mass spectrometry; Intact protein analysis

Improved detection of phosphopeptides by negative ion matrix-assisted laser desorption/ionization mass spectrometry using a proton sponge co-matrix by Shu Zhang; Zhong-Ping Yao (pp. 77-82).

Display Omitted► Use of ATT/DMAN/DHC matrix for detection of phosphopeptides by negative MALDI-MS. ► Lower limit of detection. ► Reduced signal suppression effects. ► Improved position-to-position reproducibility.Analysis of phosphopeptides is an important task in proteomic studies. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a technique very commonly used for such a purpose. Analysis of phosphopeptides by MALDI-MS is, however, still a challenging task due to the low ionization efficiency of phosphopeptides. In this study, we reported that by using a proton sponge 1,8-bis(dimethyl-amino)naphthalene (DMAN) as a co-matrix, detection of phosphopeptides by negative ion MALDI-MS could be greatly improved. Combination of DMAN with another matrix 6-aza-2-thiothymine (ATT) and additive diammonium hydrogen citrate (DHC) allowed much lower limit of detection, significantly reduced signal suppression effects and improved position-to-position reproducibility for detection of phosphopeptides by negative ion MALDI-MS. Potential applications of the matrix system in qualification of phosphopeptides and analysis of proteolytic digests of phosphorylated proteins were also demonstrated in this study.

Keywords: Phosphopeptides; MALDI; Mass spectrometry; Matrix; Proton sponge; Negative ion

Preparation of a novel molecularly imprinted polymer by the sol–gel process for sensing creatinine by Ta-Jen Li; Po-Yen Chen; Po-Chin Nien; Chia-Yu Lin; R. Vittal; Tzong-Rong Ling; Kuo-Chuan Ho (pp. 83-90).

Display Omitted► A novel molecularly imprinted polymer for creatinine (MIP_Cre) is prepared by using tetraethoxysilane and aluminum ion. ► The imprinting efficiency of MIP_Cre is confirmed in the Cre adsorption experiments. ► The MIP_Cre possesses excellent selectivities toward creatine (Cn), N-hydroxysuccinimide (NHS), andl-tyrosine (l-tyr). ► It is the Lewis acid sites rather than the silanol groups that mainly contribute to adsorbed amount of Cre by MIP_Cre.A novel molecularly imprinted polymer (MIP) was prepared and used as an artificial receptor for creatinine (Cre). A sol–gel process was used to prepare the MIP. Tetraethoxysilane (TEOS) was employed as the crosslinker for the formation of a silica matrix for the MIP. Aluminum ion (Al³⁺) was chosen as the dopant to generate Lewis acid sites in the silica matrix for interactions with Cre. Through the sol–gel process, a polymeric matrix with memory sites for Cre was obtained, and this is mentioned here as the molecularly imprinted polymer for creatinine (MIP_Cre). The imprinting efficiency of MIP_Cre was evaluated by contrasting the adsorbed amount of Cre by MIP_Cre with that by the corresponding non-imprinted polymer (NIP). Creatine (Cn), N-hydroxysuccinimide (NHS), andl-tyrosine (l-tyr) were selected as interferences to study the selectivity of the MIP_Cre. The interference studies were also conducted using binary mixtures, such as Cre/Cn, Cre/NHS, and Cre/l-tyr. All these studies reveal that the MIP_Cre possess a remarkable affinity for Cre. The crucial role of Al³⁺ in this system is discussed in detail. Furthermore, the effects of concentrations of Al³⁺ and TEOS on the adsorbed amount of Cre by MIP_Cre were also investigated.

Keywords: Aluminum ion; Creatinine; Lewis acid sites; Molecularly imprinted polymer; Silica matrix

A novel optical thrombin aptasensor based on magnetic nanoparticles and split DNAzyme by Dan Zhu; Juanjuan Luo; Xinyi Rao; Jiajia Zhang; Guifang Cheng; Pingang He; Yuzhi Fang (pp. 91-96).

Display Omitted► Label-free, simple, sensitive and cost-effective. ► A single nucleotide contains the recognition element and sensing element is utilized. ► Split DNAzyme halves is employed as sensing element, making this assays more flexible. ► This assay can detect thrombin in complex real samples due to effective magnetic separation.In this paper, we report a novel and sensitive optical sensing protocol for thrombin detection based on magnetic nanoparticles (MNPs) and thrombin aptamer, employing split HRP-mimicking DNAzyme halves as its sensing element, which can catalyze the H₂O₂-mediated oxidation of the colorless ABTS into a blue-green product. A single nucleotide containing the recognition element and sensing element is utilized in our protocol. The specific recognition of thrombin and its aptamer leads to the structure deformation of the DNA strands and causes the split of the DNAzyme halves. Therefore, the decrease of absorption spectra can be recorded by the UV–visible Spectrophotometer. DNA-coated MNPs are utilized to separate the interferential materials from the analyst, thus making this assay can be applied in the detection of thrombin in complex samples, such as human plasma. This original, sensitive and cost-effective assay showed favorable recognition for thrombin. The absorbance signals with the concentration of thrombin over a range from 0.5 to 20nM and the detection limit of thrombin was 0.5nM. The controlled experiments showed that thrombin signal was not interfered in the presence of other co-existence proteins.

Keywords: Aptamer; Thrombin; Magnetic nanoparticles (MNPs); DNAzyme

High-performance liquid chromatographic method to evaluate the hydrogen atom transfer during reaction between 1,1-diphenyl-2-picryl-hydrazyl radical and antioxidants by Ariane Boudier; Juliana Tournebize; Grzegorz Bartosz; Safae El Hani; Rachid Bengueddour; Anne Sapin-Minet; Pierre Leroy (pp. 97-106).

Display Omitted► Both 1,1-diphenyl-2-picrylhydrazyl radical and its product measurement by HPLC. ► Lowest limit of detection by monitoring 1,1-diphenyl-2-picryl-hydrazine. ► Adsorption problem of the radical on HPLC parts have been pointed out.1,1-Diphenyl-2-picrylhydrazyl (DPPH) is a stable nitrogen centred radical widely used to evaluate direct radical scavenging properties of various synthetic or natural antioxidants (AOs). The bleaching rate of DPPH absorbance at 515nm is usually monitored for this purpose. In order to avoid the interference of complex coloured natural products used as antioxidant supplements or cosmetics, HPLC systems have been reported as alternative techniques to spectrophotometry. They also rely upon measurement of DPPH quenching rate and none of them permits to identify and measure 1,1-diphenyl-2-picryl-hydrazine (DPPH-H), the reduced product of DPPH resulting from hydrogen atom transfer (HAT), which is the main mechanism of the reaction between DPPH and AOs. We presently report an HPLC method devoted to the simultaneous measurement of DPPH and DPPH-H. Both were fully separated on a C18 column eluted with acetonitrile–10mM ammonium citrate buffer pH 6.8 (70:30, v/v) and detected at 330nm. Adsorption process of DPPH onto materials of the HPLC system was pointed out. Consequently, the linearity range observed for DPPH was restricted, thus a much lower limit of detection was obtained for DPPH-H than for DPPH using standards (0.02 and 14μM, respectively). The method was applied to three commonly used AOs, i.e. Trolox^®, ascorbic acid and GSH, and compared with spectrophotometry. Further application to complex matrices (cell culture media, vegetal extracts) and nanomaterials demonstrated (i) its usefulness because of higher selectivity than colorimetry, and (ii) its help to investigate the mechanisms occurring with the free radical.

Keywords: 1,1-Diphenyl-2-picrylhydrazyl; 1,1-Diphenyl-2-picrylhydrazine; HPLC; Antioxidants; Complex matrices; Nanoparticles

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Analytica Chimica Acta (v.711, #)