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Graphene based soft nanoreactors for facile “one-step” glycan enrichment and derivatization for MALDI-TOF-MS analysis
17 September 2013,
09:36:31
Publication date: 15 December
2013
Source:Talanta, Volume 117
Author(s): Haihong Bai , Yiting Pan , Wei Tong , Wanjun Zhang , Xiaojun Ren , Fang Tian , Bo Peng , Xin Wang , Yangjun Zhang , Yulin Deng , Weijie Qin , Xiaohong Qian
Protein glycosylation is involved in the control of many important biological processes and structural alterations of the N-linked glycans are correlated with various kinds of disease. High-throughput N-glycan profiling is a key technique for elucidating the functions of glycans in biological process and disease development as well as discovering new diagnostic biomarkers. However, the low abundance of glycans existing in living organism, the competition/suppression effect of other highly abundant biological molecules and the inherent lack of alkalinity and hydrophobicity of glycans leads to particularly poor detection sensitivity in MS analysis. Here, we demonstrated the first “one-step” approach for highly efficient glycan enrichment and derivatization using reduced graphene oxide as nanoreactors and 1-pyrenebutyric hydrazide for glycan capture and derivatization, which resulted in a 33-fold increase in the glycan detection sensitivity in MALDI-TOF-MS and the identification of 48N-glycoforms from human plasma.
Source:Talanta, Volume 117
Author(s): Haihong Bai , Yiting Pan , Wei Tong , Wanjun Zhang , Xiaojun Ren , Fang Tian , Bo Peng , Xin Wang , Yangjun Zhang , Yulin Deng , Weijie Qin , Xiaohong Qian
Protein glycosylation is involved in the control of many important biological processes and structural alterations of the N-linked glycans are correlated with various kinds of disease. High-throughput N-glycan profiling is a key technique for elucidating the functions of glycans in biological process and disease development as well as discovering new diagnostic biomarkers. However, the low abundance of glycans existing in living organism, the competition/suppression effect of other highly abundant biological molecules and the inherent lack of alkalinity and hydrophobicity of glycans leads to particularly poor detection sensitivity in MS analysis. Here, we demonstrated the first “one-step” approach for highly efficient glycan enrichment and derivatization using reduced graphene oxide as nanoreactors and 1-pyrenebutyric hydrazide for glycan capture and derivatization, which resulted in a 33-fold increase in the glycan detection sensitivity in MALDI-TOF-MS and the identification of 48N-glycoforms from human plasma.
On-line speciation analysis of inorganic arsenic in complex environmental aqueous samples by pervaporation sequential injection analysis
17 September 2013,
09:36:31
Publication date: 15 December
2013
Source:Talanta, Volume 117
Author(s): Warunya Boonjob , Manuel Miró , Spas D. Kolev
A proof of concept of a novel pervaporation sequential injection (PSI) analysis method for automatic non-chromatographic speciation analysis of inorganic arsenic in complex aqueous samples is presented. The method is based on hydride generation of arsine followed by its on-line pervaporation-based membrane separation and CCD spectrophotometric detection. The concentrations of arsenite (As(III)) and arsenate (As(V)) are determined sequentially in a single sample zone. The leading section of the sample zone merges with a citric acid/citrate buffer solution (pH 4.5) for the selective reduction of As(III) to arsine while the trailing section of the sample zone merges with hydrochloric acid solution to allow the reduction of both As(III) and As(V) to arsine at pH lower than 1. Virtually identical analytical sensitivity is obtained for both As(III) and As(V) at this high acidity. The flow analyzer also accommodates in-line pH detector for monitoring of the acidity throughout the sample zone prior to hydride generation. Under optimal conditions the proposed PSI method is characterized by a limit of detection, linear calibration range and repeatability for As(III) of 22μgL−1 (3sblank level criterion), 50–1000μgL−1 and 3.0% at the 500μgL−1 level and for As(V) of 51μgL−1, 100–2000μgL−1 and 2.6% at the 500μgL−1 level, respectively. The method was validated with mixed As(III)/As(V) standard aqueous solutions and successfully applied to the determination of As(III) and As(V) in river water samples with elevated content of dissolved organic carbon and suspended particulate matter with no prior sample pretreatment. Excellent relative recoveries ranging from 98% to 104% were obtained for both As(III) and As(V).
Source:Talanta, Volume 117
Author(s): Warunya Boonjob , Manuel Miró , Spas D. Kolev
A proof of concept of a novel pervaporation sequential injection (PSI) analysis method for automatic non-chromatographic speciation analysis of inorganic arsenic in complex aqueous samples is presented. The method is based on hydride generation of arsine followed by its on-line pervaporation-based membrane separation and CCD spectrophotometric detection. The concentrations of arsenite (As(III)) and arsenate (As(V)) are determined sequentially in a single sample zone. The leading section of the sample zone merges with a citric acid/citrate buffer solution (pH 4.5) for the selective reduction of As(III) to arsine while the trailing section of the sample zone merges with hydrochloric acid solution to allow the reduction of both As(III) and As(V) to arsine at pH lower than 1. Virtually identical analytical sensitivity is obtained for both As(III) and As(V) at this high acidity. The flow analyzer also accommodates in-line pH detector for monitoring of the acidity throughout the sample zone prior to hydride generation. Under optimal conditions the proposed PSI method is characterized by a limit of detection, linear calibration range and repeatability for As(III) of 22μgL−1 (3sblank level criterion), 50–1000μgL−1 and 3.0% at the 500μgL−1 level and for As(V) of 51μgL−1, 100–2000μgL−1 and 2.6% at the 500μgL−1 level, respectively. The method was validated with mixed As(III)/As(V) standard aqueous solutions and successfully applied to the determination of As(III) and As(V) in river water samples with elevated content of dissolved organic carbon and suspended particulate matter with no prior sample pretreatment. Excellent relative recoveries ranging from 98% to 104% were obtained for both As(III) and As(V).
Application of high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC–ICP-MS) for determination of chromium compounds in the air at the workplace
17 September 2013,
09:36:31
Publication date: 15 December
2013
Source:Talanta, Volume 117
Author(s): Magdalena Stanislawska , Beata Janasik , Wojciech Wasowicz
The toxicity and bioavailability of chromium species are highly dependable on the form or species, therefore determination of total chromium is insufficient for a complete toxicological evaluation and risk assessment. An analytical method for determination of soluble and insoluble Cr (III) and Cr (VI) compounds in welding fume at workplace air has been developed. The total chromium (Cr) was determined by using quadruple inductively coupled plasma mass spectrometry (ICP-MS) equipped with a dynamic reaction cell (DRC®). Soluble trivalent and hexavalent chromium compounds were determined by high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC–ICP-MS). A high-speed, reversed-phase CR C8 column (PerkinElmer, Inc., Shelton, CT, USA) was used for the speciation of soluble Cr (III) and soluble Cr (VI). The separation was accomplished by interaction of the chromium species with the different components of the mobile phase. Cr (III) formed a complex with EDTA, i.e. retained on the column, while Cr (VI) existed in the solutions as dichromate. Alkaline extraction (2% KOH and 3% Na2CO3) and anion exchange column (PRP-X100, PEEK, Hamilton) were used for the separation of the total Cr (VI). The results of the determination of Cr (VI) were confirmed by the analysis of the certified reference material BCR CRM 545 (Cr (VI) in welding dust). The results obtained for the certified material (40.2±0.6gkg−1) and the values recorded in the examined samples (40.7±0.6gkg−1) were highly consistent. This analytical method was applied for the determination of chromium in the samples in the workplace air collected onto glass (Whatman, Ø 37mm) and membrane filters (Sartorius, 0.8μm, Ø 37mm). High performance liquid chromatography with inductively coupled plasma mass spectrometry is a remarkably powerful and versatile technique for determination of chromium species in welding fume at workplace air.
Source:Talanta, Volume 117
Author(s): Magdalena Stanislawska , Beata Janasik , Wojciech Wasowicz
The toxicity and bioavailability of chromium species are highly dependable on the form or species, therefore determination of total chromium is insufficient for a complete toxicological evaluation and risk assessment. An analytical method for determination of soluble and insoluble Cr (III) and Cr (VI) compounds in welding fume at workplace air has been developed. The total chromium (Cr) was determined by using quadruple inductively coupled plasma mass spectrometry (ICP-MS) equipped with a dynamic reaction cell (DRC®). Soluble trivalent and hexavalent chromium compounds were determined by high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC–ICP-MS). A high-speed, reversed-phase CR C8 column (PerkinElmer, Inc., Shelton, CT, USA) was used for the speciation of soluble Cr (III) and soluble Cr (VI). The separation was accomplished by interaction of the chromium species with the different components of the mobile phase. Cr (III) formed a complex with EDTA, i.e. retained on the column, while Cr (VI) existed in the solutions as dichromate. Alkaline extraction (2% KOH and 3% Na2CO3) and anion exchange column (PRP-X100, PEEK, Hamilton) were used for the separation of the total Cr (VI). The results of the determination of Cr (VI) were confirmed by the analysis of the certified reference material BCR CRM 545 (Cr (VI) in welding dust). The results obtained for the certified material (40.2±0.6gkg−1) and the values recorded in the examined samples (40.7±0.6gkg−1) were highly consistent. This analytical method was applied for the determination of chromium in the samples in the workplace air collected onto glass (Whatman, Ø 37mm) and membrane filters (Sartorius, 0.8μm, Ø 37mm). High performance liquid chromatography with inductively coupled plasma mass spectrometry is a remarkably powerful and versatile technique for determination of chromium species in welding fume at workplace air.
Bromination of aromatic compounds by residual bromide in sodium chloride matrix modifier salt during heated headspace GC/MS analysis
17 September 2013,
09:36:31
Publication date: 15 December
2013
Source:Talanta, Volume 117
Author(s): Dennis D. Fine , Saebom Ko , Scott Huling
Analytical artifacts attributed to the bromination of toluene, xylenes, and trimethylbenzenes were found during the heated headspace gas chromatography/mass spectrometry (GC/MS) analysis of aqueous samples. The aqueous samples were produced from Fenton-like chemical oxidation reactions and contained aromatic compounds, hydrogen peroxide (H2O2), and ferric sulfate. Prior to GC/MS headspace analysis, the samples were acidified (pH<2), and sodium chloride was amended to the headspace vial as a matrix modifier. The brominated artifacts were generated during heated headspace analysis. Further, when samples were spiked with a mixture of volatile chlorinated and aromatic compounds (50µg/L), poor spike recoveries of toluene and xylenes occurred, and in some cases complete loss of trimethylbenzenes and naphthalene resulted. Where poor recovery of aromatic spike compounds occurred, brominated aromatic compounds were found. The only significant source of bromine in the reaction scheme is the bromide typically present (<0.01% w/w) in the sodium chloride amended to the samples. Conversely, brominated artifacts were absent when a buffered salt mixture composed of sodium chloride and potassium phosphate dibasic/monobasic was used as a matrix modifier and raised the sample pH (pH~6). This indicated that the brominated artifacts resulted from the reaction of the aromatic compounds with BrCl, which was formed by the reaction of H2O2, chloride, and bromide under acidic conditions. An alternative matrix modifier salt is recommended that prevents the bromination reaction and avoids these deleterious effects on sample integrity during headspace analysis.
Source:Talanta, Volume 117
Author(s): Dennis D. Fine , Saebom Ko , Scott Huling
Analytical artifacts attributed to the bromination of toluene, xylenes, and trimethylbenzenes were found during the heated headspace gas chromatography/mass spectrometry (GC/MS) analysis of aqueous samples. The aqueous samples were produced from Fenton-like chemical oxidation reactions and contained aromatic compounds, hydrogen peroxide (H2O2), and ferric sulfate. Prior to GC/MS headspace analysis, the samples were acidified (pH<2), and sodium chloride was amended to the headspace vial as a matrix modifier. The brominated artifacts were generated during heated headspace analysis. Further, when samples were spiked with a mixture of volatile chlorinated and aromatic compounds (50µg/L), poor spike recoveries of toluene and xylenes occurred, and in some cases complete loss of trimethylbenzenes and naphthalene resulted. Where poor recovery of aromatic spike compounds occurred, brominated aromatic compounds were found. The only significant source of bromine in the reaction scheme is the bromide typically present (<0.01% w/w) in the sodium chloride amended to the samples. Conversely, brominated artifacts were absent when a buffered salt mixture composed of sodium chloride and potassium phosphate dibasic/monobasic was used as a matrix modifier and raised the sample pH (pH~6). This indicated that the brominated artifacts resulted from the reaction of the aromatic compounds with BrCl, which was formed by the reaction of H2O2, chloride, and bromide under acidic conditions. An alternative matrix modifier salt is recommended that prevents the bromination reaction and avoids these deleterious effects on sample integrity during headspace analysis.
Concerted derivatization and concentration method with dispersive liquid–liquid microextraction for liquid chromatographic analysis of 5-hydroxyindoles in human serum
17 September 2013,
09:36:31
Publication date: 15 December
2013
Source:Talanta, Volume 117
Author(s): Tadashi Hayama , Yurika Yabuuchi , Tomomi Iwamatsu , Erina Tamashima , Yusuke Kawami , Miki Itoyama , Hideyuki Yoshida , Masatoshi Yamaguchi , Hitoshi Nohta
We developed a concerted derivatization and concentration method based on dispersive liquid–liquid microextraction (DLLME) for the liquid chromatography (LC) determination of 5-hydroxyindoles (5-HIs; serotonin, 5-hydroxyindole-3-acetic acid, N-acetylserotonin, and 5-hydroxytryptohol). Concerted derivatization and concentration could be affected by adding a mixture of an ionic liquid (1-hexyl-3-methylimidazolium hexafluorophosphate, extraction solvent), methanol (disperser), and water containing fluorescence derivatization reagents [benzylamine and potassium hexacyanoferrate(III)] into the sample. The resulting sedimented phase was injected into a reversed-phase LC column using a mixture of acetonitrile and 250mM acetate buffer (pH 4.3) as the mobile phase for gradient elution, and the derivatives obtained were fluorometrically detected at excitation and emission wavelengths of 345nm and 452nm, respectively. The derivatization (reagent concentrations and pH) and extraction (extraction and disperser solvent type) conditions were optimized simultaneously. The limits of detection of the 5-HIs were in the range of 0.08–0.33nM. The method was validated for 10 and 50pmol/mL human serum levels, and the recovery of 5-HIs was between 66% and 98%, within a relative standard deviation of 9.5%. The proposed method is well suited for the highly sensitive analysis of trace amounts of 5-HIs in human serum samples.
Source:Talanta, Volume 117
Author(s): Tadashi Hayama , Yurika Yabuuchi , Tomomi Iwamatsu , Erina Tamashima , Yusuke Kawami , Miki Itoyama , Hideyuki Yoshida , Masatoshi Yamaguchi , Hitoshi Nohta
We developed a concerted derivatization and concentration method based on dispersive liquid–liquid microextraction (DLLME) for the liquid chromatography (LC) determination of 5-hydroxyindoles (5-HIs; serotonin, 5-hydroxyindole-3-acetic acid, N-acetylserotonin, and 5-hydroxytryptohol). Concerted derivatization and concentration could be affected by adding a mixture of an ionic liquid (1-hexyl-3-methylimidazolium hexafluorophosphate, extraction solvent), methanol (disperser), and water containing fluorescence derivatization reagents [benzylamine and potassium hexacyanoferrate(III)] into the sample. The resulting sedimented phase was injected into a reversed-phase LC column using a mixture of acetonitrile and 250mM acetate buffer (pH 4.3) as the mobile phase for gradient elution, and the derivatives obtained were fluorometrically detected at excitation and emission wavelengths of 345nm and 452nm, respectively. The derivatization (reagent concentrations and pH) and extraction (extraction and disperser solvent type) conditions were optimized simultaneously. The limits of detection of the 5-HIs were in the range of 0.08–0.33nM. The method was validated for 10 and 50pmol/mL human serum levels, and the recovery of 5-HIs was between 66% and 98%, within a relative standard deviation of 9.5%. The proposed method is well suited for the highly sensitive analysis of trace amounts of 5-HIs in human serum samples.
Graphical abstract
Novel extraction induced by emulsion breaking as a tool for the determination of trace concentrations of Cu, Mn and Ni in biodiesel by electrothermal atomic absorption spectrometry
17 September 2013,
09:36:31
Publication date: 15 December
2013
Source:Talanta, Volume 117
Author(s): Fernanda M. Pereira , Renata C. Zimpeck , Daniel M. Brum , Ricardo J. Cassella
This work proposes a novel method for the determination of trace concentrations of Cu, Mn and Ni in biodiesel samples by electrothermal atomic absorption spectrometry. In order to overcome problems related to the organic matrix in the direct introduction of the samples, a new extraction approach was investigated. The method was based on the extraction induced by emulsion breaking, in which metals were transferred from the biodiesel to an acid aqueous phase after formation and breaking of a water-in-oil emulsion prepared by mixing the biodiesel sample with an aqueous solution containing surfactant and nitric acid. Several parameters that could influence the performance of the system were evaluated. Quantitative extractions of the analytes were obtained when the extraction was performed using an emulsifier solution containing 2.1molL−1 of HNO3 and 7% m/v of Triton X-100. The extraction time had remarkable influence on the efficiency of the process, being necessary an agitation time of 60min to achieve maximum extraction. The limits of quantification were below 1µgL−1 for the three analytes under study. The accuracy of the method was tested by application of a recovery test (recovery percentages between 89% and 109% were observed) and by comparison with a well-established method, taken as reference.
Source:Talanta, Volume 117
Author(s): Fernanda M. Pereira , Renata C. Zimpeck , Daniel M. Brum , Ricardo J. Cassella
This work proposes a novel method for the determination of trace concentrations of Cu, Mn and Ni in biodiesel samples by electrothermal atomic absorption spectrometry. In order to overcome problems related to the organic matrix in the direct introduction of the samples, a new extraction approach was investigated. The method was based on the extraction induced by emulsion breaking, in which metals were transferred from the biodiesel to an acid aqueous phase after formation and breaking of a water-in-oil emulsion prepared by mixing the biodiesel sample with an aqueous solution containing surfactant and nitric acid. Several parameters that could influence the performance of the system were evaluated. Quantitative extractions of the analytes were obtained when the extraction was performed using an emulsifier solution containing 2.1molL−1 of HNO3 and 7% m/v of Triton X-100. The extraction time had remarkable influence on the efficiency of the process, being necessary an agitation time of 60min to achieve maximum extraction. The limits of quantification were below 1µgL−1 for the three analytes under study. The accuracy of the method was tested by application of a recovery test (recovery percentages between 89% and 109% were observed) and by comparison with a well-established method, taken as reference.
Stir bar sorptive extraction coupled to liquid chromatography-tandem mass spectrometry for the determination of pesticides in water samples: Method validation and measurement uncertainty
17 September 2013,
09:36:31
Publication date: 15 November
2013
Source:Talanta, Volume 116
Author(s): Christelle Margoum , Céline Guillemain , Xi Yang , Marina Coquery
Stir bar sorptive extraction followed by liquid desorption and high performance liquid chromatography with tandem mass spectrometry (SBSE-LD-LC-MSMS) has been developed for the determination of 15 pesticides or selected metabolites from different families (herbicides, insecticides, fungicides) in surface water samples. The optimization of parameters that could influence SBSE-LD efficiency was carried out by means of experimental design. Optimized conditions were established as follows concerning extraction time, stirring speed, aqueous medium characteristics (ionic strength and polarity) and back desorption solvent and time, respectively: 3h (800rpm), addition of 10% of sodium chloride, no addition of methanol as organic modifier, and 15min ultrasonic desorption in equivolume mixtures of acetonitrile-methanol. A specific and thorough cleanup procedure was developed and applied to each stir bar to avoid possible carry-over between consecutive extractions with the same stir bar. Pesticide quantification in water was achieved thanks to matrix matched calibration. Mean recoveries ranged from 93 to 101% (RSD <17%, n=30). Validated limits of quantification in matrix were between 0.02 and 1µgL−1, depending on the compound. A specific experimental design was conducted to evaluate the measurement uncertainty, which was comprised between 13 and 51%, whatever the pesticide and the concentration level. The applicability of the SBSE-LD-LCMSMS method was evaluated by analyzing surface water samples and by comparing with conventional solid phase extraction-LC-MSMS procedure.
Source:Talanta, Volume 116
Author(s): Christelle Margoum , Céline Guillemain , Xi Yang , Marina Coquery
Stir bar sorptive extraction followed by liquid desorption and high performance liquid chromatography with tandem mass spectrometry (SBSE-LD-LC-MSMS) has been developed for the determination of 15 pesticides or selected metabolites from different families (herbicides, insecticides, fungicides) in surface water samples. The optimization of parameters that could influence SBSE-LD efficiency was carried out by means of experimental design. Optimized conditions were established as follows concerning extraction time, stirring speed, aqueous medium characteristics (ionic strength and polarity) and back desorption solvent and time, respectively: 3h (800rpm), addition of 10% of sodium chloride, no addition of methanol as organic modifier, and 15min ultrasonic desorption in equivolume mixtures of acetonitrile-methanol. A specific and thorough cleanup procedure was developed and applied to each stir bar to avoid possible carry-over between consecutive extractions with the same stir bar. Pesticide quantification in water was achieved thanks to matrix matched calibration. Mean recoveries ranged from 93 to 101% (RSD <17%, n=30). Validated limits of quantification in matrix were between 0.02 and 1µgL−1, depending on the compound. A specific experimental design was conducted to evaluate the measurement uncertainty, which was comprised between 13 and 51%, whatever the pesticide and the concentration level. The applicability of the SBSE-LD-LCMSMS method was evaluated by analyzing surface water samples and by comparing with conventional solid phase extraction-LC-MSMS procedure.
Graphical abstract
Investigating of labelling and detection of transthyretin synthetic peptide derivatized with naphthalene-2,3-dicarboxaldehyde
17 September 2013,
09:36:31
Publication date: 15 November
2013
Source:Talanta, Volume 116
Author(s): Mathilde Faure , Sonia Korchane , Isabelle Le Potier , Antoine Pallandre , Claude Deslouis , Anne-Marie Haghiri-Gosnet , Jean Gamby
Labelling and detection of a synthetic peptide (PN) mimicking a tryptic fragment of interest for the diagnosis of familial amyloidal polyneuropathy have been investigated optically and electrochemically. We decided to covalently label naphtalene-2,3-dicarboxyaldehyde (NDA), a fluorogenic and electroactive molecule on PN. First, the optimization of the labelling chemical reaction was performed by capillary electrophoresis coupled with laser induced fluorescence detection (CE-LIF). The analytical parameters such as separation efficiency and peak area were considered to propose this optimized derivatization reaction. The results obtained allowed us to establish the pH and ionic strength of the derivatization buffer, the molar ratio between NDA and PN and the reaction time of the labelling. Optimal conditions are obtained when [NDA]/[PN]=40, buffer pH of 9, buffer ionic strength of 70mM and reaction time of 15min. Second, differential pulse voltammetry (DPV) and cyclic voltammetry (CV) were also used to characterize NDA-labelled PN and different electroinactive amino acids (histidine, lysine, serine, threonine) which are in the PN sequence. The electrochemical detection experiments demonstrated that the labelled biomolecules could be also easily detected at low concentration. Moreover, the derivatization reaction could be followed to describe more precisely the labelling process of these biomolecules. Optimal conditions for labelling are obtained when [NDA]total/[CN-] ratio =1 and [NDA]total/[amino acid or peptide]=100 with a buffer having a pH=9 on a glassy carbon electrode. In all cases, an obvious oxidation peak for the N-2-substituted-1-cyanobenz-[f]-isoindole derivative (CBI) has been observed at 0.5–0.7V/SCE. The multi-labelling of PN and lysine were shown with DPV. We presumed this result to occur because of the shouldered shape of the DPV peak shape. These experiments confirm that NDA can be used as a derivative agent for PN, allowing for electrochemical and fluorescence detections with a limit of detection of labelled PN estimated at 0.2µM and 5µM, respectively.
Source:Talanta, Volume 116
Author(s): Mathilde Faure , Sonia Korchane , Isabelle Le Potier , Antoine Pallandre , Claude Deslouis , Anne-Marie Haghiri-Gosnet , Jean Gamby
Labelling and detection of a synthetic peptide (PN) mimicking a tryptic fragment of interest for the diagnosis of familial amyloidal polyneuropathy have been investigated optically and electrochemically. We decided to covalently label naphtalene-2,3-dicarboxyaldehyde (NDA), a fluorogenic and electroactive molecule on PN. First, the optimization of the labelling chemical reaction was performed by capillary electrophoresis coupled with laser induced fluorescence detection (CE-LIF). The analytical parameters such as separation efficiency and peak area were considered to propose this optimized derivatization reaction. The results obtained allowed us to establish the pH and ionic strength of the derivatization buffer, the molar ratio between NDA and PN and the reaction time of the labelling. Optimal conditions are obtained when [NDA]/[PN]=40, buffer pH of 9, buffer ionic strength of 70mM and reaction time of 15min. Second, differential pulse voltammetry (DPV) and cyclic voltammetry (CV) were also used to characterize NDA-labelled PN and different electroinactive amino acids (histidine, lysine, serine, threonine) which are in the PN sequence. The electrochemical detection experiments demonstrated that the labelled biomolecules could be also easily detected at low concentration. Moreover, the derivatization reaction could be followed to describe more precisely the labelling process of these biomolecules. Optimal conditions for labelling are obtained when [NDA]total/[CN-] ratio =1 and [NDA]total/[amino acid or peptide]=100 with a buffer having a pH=9 on a glassy carbon electrode. In all cases, an obvious oxidation peak for the N-2-substituted-1-cyanobenz-[f]-isoindole derivative (CBI) has been observed at 0.5–0.7V/SCE. The multi-labelling of PN and lysine were shown with DPV. We presumed this result to occur because of the shouldered shape of the DPV peak shape. These experiments confirm that NDA can be used as a derivative agent for PN, allowing for electrochemical and fluorescence detections with a limit of detection of labelled PN estimated at 0.2µM and 5µM, respectively.
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