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Analytical and Bioanalytical Chemistry (v.383, #2)
Simultaneous multi-species determination of trimethyllead, monomethylmercury and three butyltin compounds by species-specific isotope dilution GC–ICP–MS in biological samples by Nataliya Poperechna; Klaus G. Heumann (pp. 153-159).
An accurate and sensitive multi-species species-specific isotope dilution GC–ICP–MS method was developed for the simultaneous determination of trimethyllead (Me3Pb+), monomethylmercury (MeHg+) and the three butyltin species Bu3Sn+, Bu2Sn2+, and BuSn3+ in biological samples. The method was validated by three biological reference materials (CRM 477, mussel tissue certified for butyltins; CRM 463, tuna fish certified for MeHg+; DORM 2, dogfish muscle certified for MeHg+). Under certain conditions, and with minor modifications of the sample pretreatment procedure, this method could also be transferred to environmental samples such as sediments, as demonstrated by analyzing sediment reference material BCR 646 (freshwater sediment, certified for butyltins). The detection limits of the multi-species GC–ICP–IDMS method for biological samples were 1.4 ng g−1 for MeHg+, 0.06 ng g−1 for Me3Pb+, 0.3 ng g−1 for BuSn3+ and Bu3Sn+, and 1.2 ng g−1 for Bu2Sn2+. Because of the high relevance of these heavy metal alkyl species to the quality assurance of seafood, the method was also applied to corresponding samples purchased from a supermarket. The methylated lead fraction in these samples, correlated to total lead, varied over a broad range (from 0.01% to 7.6%). On the other hand, the MeHg+ fraction was much higher, normally in the range of 80–100%. Considering that we may expect tighter legislative limitations on MeHg+ levels in seafood in the future, we found the highest methylmercury contents (up to 10.6 μg g−1) in two shark samples, an animal which is at the end of the marine food chain, whereas MeHg+ contents of less than 0.2 μg g−1 were found in most other seafood samples; these results correlate with the idea that MeHg+ is usually of biological origin in the marine environment. The concentration of butyltins and the fraction of the total tin content that is from butyltins strongly depend on possible contamination, due to the exclusively anthropogenic character of these compounds. A broad variation in the butylated tin fraction (in the range of <0.3–49%) was therefore observed in different seafood samples. Corresponding isotope-labeled spike compounds (except for trimethyllead) are commercially available for all of these compounds, and since these can be used in the multi-species species-specific GC-ICP-IDMS method developed here, this technique shows great potential for routine analysis in the future.
Keywords: Simultaneous multi-species determination; Methylated mercury and lead; Butyltins; Species-specific isotope dilution technique; GC–ICP–MS coupling; Seafood
Simultaneous multi-species determination of trimethyllead, monomethylmercury and three butyltin compounds by species-specific isotope dilution GC–ICP–MS in biological samples by Nataliya Poperechna; Klaus G. Heumann (pp. 153-159).
An accurate and sensitive multi-species species-specific isotope dilution GC–ICP–MS method was developed for the simultaneous determination of trimethyllead (Me3Pb+), monomethylmercury (MeHg+) and the three butyltin species Bu3Sn+, Bu2Sn2+, and BuSn3+ in biological samples. The method was validated by three biological reference materials (CRM 477, mussel tissue certified for butyltins; CRM 463, tuna fish certified for MeHg+; DORM 2, dogfish muscle certified for MeHg+). Under certain conditions, and with minor modifications of the sample pretreatment procedure, this method could also be transferred to environmental samples such as sediments, as demonstrated by analyzing sediment reference material BCR 646 (freshwater sediment, certified for butyltins). The detection limits of the multi-species GC–ICP–IDMS method for biological samples were 1.4 ng g−1 for MeHg+, 0.06 ng g−1 for Me3Pb+, 0.3 ng g−1 for BuSn3+ and Bu3Sn+, and 1.2 ng g−1 for Bu2Sn2+. Because of the high relevance of these heavy metal alkyl species to the quality assurance of seafood, the method was also applied to corresponding samples purchased from a supermarket. The methylated lead fraction in these samples, correlated to total lead, varied over a broad range (from 0.01% to 7.6%). On the other hand, the MeHg+ fraction was much higher, normally in the range of 80–100%. Considering that we may expect tighter legislative limitations on MeHg+ levels in seafood in the future, we found the highest methylmercury contents (up to 10.6 μg g−1) in two shark samples, an animal which is at the end of the marine food chain, whereas MeHg+ contents of less than 0.2 μg g−1 were found in most other seafood samples; these results correlate with the idea that MeHg+ is usually of biological origin in the marine environment. The concentration of butyltins and the fraction of the total tin content that is from butyltins strongly depend on possible contamination, due to the exclusively anthropogenic character of these compounds. A broad variation in the butylated tin fraction (in the range of <0.3–49%) was therefore observed in different seafood samples. Corresponding isotope-labeled spike compounds (except for trimethyllead) are commercially available for all of these compounds, and since these can be used in the multi-species species-specific GC-ICP-IDMS method developed here, this technique shows great potential for routine analysis in the future.
Keywords: Simultaneous multi-species determination; Methylated mercury and lead; Butyltins; Species-specific isotope dilution technique; GC–ICP–MS coupling; Seafood
Use of the three-phase model and headspace analysis for the facile determination of all partition/association constants for highly volatile solute–cyclodextrin–water systems by Andrew W. Lantz; Sean M. Wetterer; Daniel W. Armstrong (pp. 160-166).
A versatile method for measuring the partition coefficients of volatile analytes with an aqueous pseudophase using headspace gas chromatography is reported. A “three-phase” model accounts for all equilibria present in the system, including the partitioning of the analyte in the gas and aqueous phases to the pseudophase. This method is applicable to a wide variety of volatile analytes and aqueous pseudophases, providing that sufficient pseudophase may be used to reduce the analyte partial pressure. Generally, the method offers good reproducibility and high sensitivity. The associations of five volatile analytes (hydrogen sulfide, methanethiol, dimethyl sulfide, dichloromethane, and ethyl ether) with various cyclodextrins were examined. All analytes were found to partition preferentially to the cyclodextrin pseudophase compared to the aqueous phase. In addition, several analyte–cyclodextrin combinations formed insoluble complexes in solution that enhanced the extraction of the analyte from the gas and aqueous phases. Derivatization of the cyclodextrins generally decreased the extent of analyte–cyclodextrin interaction.
Keywords: Henry's Law; Volatiles; Partition coefficient; Headspace gas chromatography; Pseudophase
Use of the three-phase model and headspace analysis for the facile determination of all partition/association constants for highly volatile solute–cyclodextrin–water systems by Andrew W. Lantz; Sean M. Wetterer; Daniel W. Armstrong (pp. 160-166).
A versatile method for measuring the partition coefficients of volatile analytes with an aqueous pseudophase using headspace gas chromatography is reported. A “three-phase” model accounts for all equilibria present in the system, including the partitioning of the analyte in the gas and aqueous phases to the pseudophase. This method is applicable to a wide variety of volatile analytes and aqueous pseudophases, providing that sufficient pseudophase may be used to reduce the analyte partial pressure. Generally, the method offers good reproducibility and high sensitivity. The associations of five volatile analytes (hydrogen sulfide, methanethiol, dimethyl sulfide, dichloromethane, and ethyl ether) with various cyclodextrins were examined. All analytes were found to partition preferentially to the cyclodextrin pseudophase compared to the aqueous phase. In addition, several analyte–cyclodextrin combinations formed insoluble complexes in solution that enhanced the extraction of the analyte from the gas and aqueous phases. Derivatization of the cyclodextrins generally decreased the extent of analyte–cyclodextrin interaction.
Keywords: Henry's Law; Volatiles; Partition coefficient; Headspace gas chromatography; Pseudophase
Determination of chromium in whole blood by DRC–ICP–MS: spectral and non-spectral interferences by C. Bonnefoy; A. Menudier; C. Moesch; G. Lachâtre; J. -M. Mermet (pp. 167-173).
Quantification of chromium in whole blood has been performed by ICP–quadrupole MS. The spectrometer was equipped with a dynamic reaction cell (DRC) with ammonia as reaction gas. The rejection parameter q (RPq) of the DRC and the flow rate of ammonia (NH3) were optimized and set at 0.7 and 0.6 mL min−1, respectively. Blood was diluted 1:51 (v/v) with an aqueous solution containing 0.1 mg L−1 NH4OH, 0.1 g L−1 EDTA, 5 mg L−1 n-butanol, and 0.1‰ Triton X100. Non-spectral matrix effects observed when using the DRC were confirmed by use of vanadium. External calibration with blank and standard solutions prepared in purified water led to biased results for quality control samples. Standard addition calibration was therefore used and its validity verified. By comparing the slopes and calculating residues, it was proved that the plot obtained with standard additions and the plot obtained from blood samples of different concentrations were aligned down to 0.05 μg L−1 after dilution.
Keywords: Inductively coupled plasma–mass spectrometry; Dynamic reaction cell; Chromium; Whole blood; Standard additions; Matrix effects
Determination of chromium in whole blood by DRC–ICP–MS: spectral and non-spectral interferences by C. Bonnefoy; A. Menudier; C. Moesch; G. Lachâtre; J. -M. Mermet (pp. 167-173).
Quantification of chromium in whole blood has been performed by ICP–quadrupole MS. The spectrometer was equipped with a dynamic reaction cell (DRC) with ammonia as reaction gas. The rejection parameter q (RPq) of the DRC and the flow rate of ammonia (NH3) were optimized and set at 0.7 and 0.6 mL min−1, respectively. Blood was diluted 1:51 (v/v) with an aqueous solution containing 0.1 mg L−1 NH4OH, 0.1 g L−1 EDTA, 5 mg L−1 n-butanol, and 0.1‰ Triton X100. Non-spectral matrix effects observed when using the DRC were confirmed by use of vanadium. External calibration with blank and standard solutions prepared in purified water led to biased results for quality control samples. Standard addition calibration was therefore used and its validity verified. By comparing the slopes and calculating residues, it was proved that the plot obtained with standard additions and the plot obtained from blood samples of different concentrations were aligned down to 0.05 μg L−1 after dilution.
Keywords: Inductively coupled plasma–mass spectrometry; Dynamic reaction cell; Chromium; Whole blood; Standard additions; Matrix effects
Troubleshooting with cell blanks in PLE extraction by V. Fernández-González; G. Grueiro-Noche; E. Concha-Graña; M. I. Turnes-Carou; S. Muniategui-Lorenzo; P. López-Mahía; D. Prada-Rodríguez (pp. 174-181).
The blank extracts obtained from the pressurized liquid extraction (PLE) of a 11 mL empty cell of ASE 200 were analysed by GC-FID and GC-ECD and many interfering peaks were detected, which could difficult the trace analysis of persistent organic pollutants (i.e. polycyclic aromatic hydrocarbons, aliphatic hydrocarbons and organochlorine pesticides). These interfering compounds were identified as phthalates, silicones and organic acids and their sources were established. A solution to this analytical trouble is a previous extraction step of the empty cell under the same conditions optimised for the sample extraction.
Keywords: Pressurized solvent extraction; PLE; ASE cell blanks
Troubleshooting with cell blanks in PLE extraction by V. Fernández-González; G. Grueiro-Noche; E. Concha-Graña; M. I. Turnes-Carou; S. Muniategui-Lorenzo; P. López-Mahía; D. Prada-Rodríguez (pp. 174-181).
The blank extracts obtained from the pressurized liquid extraction (PLE) of a 11 mL empty cell of ASE 200 were analysed by GC-FID and GC-ECD and many interfering peaks were detected, which could difficult the trace analysis of persistent organic pollutants (i.e. polycyclic aromatic hydrocarbons, aliphatic hydrocarbons and organochlorine pesticides). These interfering compounds were identified as phthalates, silicones and organic acids and their sources were established. A solution to this analytical trouble is a previous extraction step of the empty cell under the same conditions optimised for the sample extraction.
Keywords: Pressurized solvent extraction; PLE; ASE cell blanks
A fluorimetric assay for cortisol by Daniel Appel; Rolf D. Schmid; Calin-Aurel Dragan; Matthias Bureik; Vlada B. Urlacher (pp. 182-186).
A simple, rapid and sensitive fluorimetric assay for the quantitative determination of cortisol is reported. The assay is based on the formation of a fluorescent dye when cortisol is incubated with a mixture of sulfuric acid and acetic acid. The fluorescence spectrum recorded for the resulting dye shows a maximum extinction at 475 nm and a maximum emission at 525 nm. The solvent 2-methyl-4-pentanone was used for extraction and was found to act as a fluorescence amplifier. A limit of detection of 2.7 μM was achieved, making it possible to forego solvent evaporation. The assay suffers minor interference from 11-deoxycortisol which exhibits low fluorescence at λ ex: 460 nm; λ em: 505 nm. Typical standard deviations were below 4%. We validated the assay using a biotransformation with recombinant Schizosaccharomyces pombe which regioselectively hydroxylates 11-deoxycortisol to cortisol. The method described herein is suitable for preliminary screening of microorganisms capable of steroid hydroxylation.
Keywords: Schizosaccharomyces pombe ; Cortisol; 11-deoxycortisol; Fluorimetric assay; Fission yeast
A fluorimetric assay for cortisol by Daniel Appel; Rolf D. Schmid; Calin-Aurel Dragan; Matthias Bureik; Vlada B. Urlacher (pp. 182-186).
A simple, rapid and sensitive fluorimetric assay for the quantitative determination of cortisol is reported. The assay is based on the formation of a fluorescent dye when cortisol is incubated with a mixture of sulfuric acid and acetic acid. The fluorescence spectrum recorded for the resulting dye shows a maximum extinction at 475 nm and a maximum emission at 525 nm. The solvent 2-methyl-4-pentanone was used for extraction and was found to act as a fluorescence amplifier. A limit of detection of 2.7 μM was achieved, making it possible to forego solvent evaporation. The assay suffers minor interference from 11-deoxycortisol which exhibits low fluorescence at λ ex: 460 nm; λ em: 505 nm. Typical standard deviations were below 4%. We validated the assay using a biotransformation with recombinant Schizosaccharomyces pombe which regioselectively hydroxylates 11-deoxycortisol to cortisol. The method described herein is suitable for preliminary screening of microorganisms capable of steroid hydroxylation.
Keywords: Schizosaccharomyces pombe ; Cortisol; 11-deoxycortisol; Fluorimetric assay; Fission yeast
Surface characterization of 3-glycidoxypropyltrimethoxysilane films on silicon-based substrates by April K. Y. Wong; Ulrich J. Krull (pp. 187-200).
Silane coupling agents are commonly used to activate surfaces for subsequent immobilization of biomolecules. The homogeneity and surface morphology of silane films is important for controlling the structural order of immobilized single-stranded DNA probes based on oligonucleotides. The surfaces of silicon wafers and glass slides with covalently attached 3-glycidoxypropyltrimethoxysilane (GOPS) have been characterized by using angularly dependent X-ray photoelectron spectroscopy (XPS), time-of-flight secondary-ion mass spectrometry (ToF–SIMS), atomic force microscopy (AFM), scanning electron microscopy (SEM), and monochromatic and spectroscopic ellipsometry. XPS and ToF–SIMS data provided evidence of complete surface coverage by GOPS. Data from angularly resolved XPS and ellipsometry methods suggested that the GOPS films were of monolayer thickness. AFM and SEM data indicated the presence of films that consisted of nodules approximately 50–100 nm in diameter. Modeling suggested that the nodules may lead to a nanoscale structural morphology that might influence the hybridization kinetics and thermodynamics of immobilized oligonucleotides.
Keywords: Glass; Silicon wafers; 3-Glycidoxypropyltrimethoxysilane; Surface analysis
Surface characterization of 3-glycidoxypropyltrimethoxysilane films on silicon-based substrates by April K. Y. Wong; Ulrich J. Krull (pp. 187-200).
Silane coupling agents are commonly used to activate surfaces for subsequent immobilization of biomolecules. The homogeneity and surface morphology of silane films is important for controlling the structural order of immobilized single-stranded DNA probes based on oligonucleotides. The surfaces of silicon wafers and glass slides with covalently attached 3-glycidoxypropyltrimethoxysilane (GOPS) have been characterized by using angularly dependent X-ray photoelectron spectroscopy (XPS), time-of-flight secondary-ion mass spectrometry (ToF–SIMS), atomic force microscopy (AFM), scanning electron microscopy (SEM), and monochromatic and spectroscopic ellipsometry. XPS and ToF–SIMS data provided evidence of complete surface coverage by GOPS. Data from angularly resolved XPS and ellipsometry methods suggested that the GOPS films were of monolayer thickness. AFM and SEM data indicated the presence of films that consisted of nodules approximately 50–100 nm in diameter. Modeling suggested that the nodules may lead to a nanoscale structural morphology that might influence the hybridization kinetics and thermodynamics of immobilized oligonucleotides.
Keywords: Glass; Silicon wafers; 3-Glycidoxypropyltrimethoxysilane; Surface analysis
Comparison and evaluation of analysis procedures for the quantification of (2-methoxyethoxy)acetic acid in urine by Clayton B’Hymer; Mary Ann Butler; Kenneth L. Cheever (pp. 201-209).
Several extraction and derivatization procedures were evaluated for the quantification of (2-methoxyethoxy)acetic acid (MEAA) in urine. MEAA is a metabolite and a biomarker for exposure to 2-(2-methoxyethoxy)ethanol, a glycol ether with widespread use in various industrial applications and the specific use as an anti-icing additive in the military jet fuel formulation JP-8. Quantification of glycol ether biomarkers is an active area of analytical research. Various sample preparation procedures were evaluated: liquid–liquid extraction (LLE) using ethyl acetate yielded the highest recovery, and solid-phase extraction (SPE) gave low recovery of MEAA. Two derivatization procedures were thoroughly investigated and validated, namely, silylation of MEAA with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA), and esterification of MEAA using ethanol. Quantification was performed by gas chromatography (GC) with a mass spectrometer as detector and using a polydimethylsiloxane (HP-1) capillary column. Deuterated 2-butoxyacetic acid (d-BAA) was used as an internal standard. Recovery studies of spiked human urine demonstrated the accuracy and precision of both procedures. The limit of detection (LOD) and other figures of merit for both derivatization procedures will be discussed in detail. Applications of these analysis procedures are also discussed.
Keywords: Glycol ethers; Alkoxyacetic acids; 2-(2-Methoxyethoxy)ethanol; Urinary biomarkers
Comparison and evaluation of analysis procedures for the quantification of (2-methoxyethoxy)acetic acid in urine by Clayton B’Hymer; Mary Ann Butler; Kenneth L. Cheever (pp. 201-209).
Several extraction and derivatization procedures were evaluated for the quantification of (2-methoxyethoxy)acetic acid (MEAA) in urine. MEAA is a metabolite and a biomarker for exposure to 2-(2-methoxyethoxy)ethanol, a glycol ether with widespread use in various industrial applications and the specific use as an anti-icing additive in the military jet fuel formulation JP-8. Quantification of glycol ether biomarkers is an active area of analytical research. Various sample preparation procedures were evaluated: liquid–liquid extraction (LLE) using ethyl acetate yielded the highest recovery, and solid-phase extraction (SPE) gave low recovery of MEAA. Two derivatization procedures were thoroughly investigated and validated, namely, silylation of MEAA with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA), and esterification of MEAA using ethanol. Quantification was performed by gas chromatography (GC) with a mass spectrometer as detector and using a polydimethylsiloxane (HP-1) capillary column. Deuterated 2-butoxyacetic acid (d-BAA) was used as an internal standard. Recovery studies of spiked human urine demonstrated the accuracy and precision of both procedures. The limit of detection (LOD) and other figures of merit for both derivatization procedures will be discussed in detail. Applications of these analysis procedures are also discussed.
Keywords: Glycol ethers; Alkoxyacetic acids; 2-(2-Methoxyethoxy)ethanol; Urinary biomarkers
Validated HPLC–MS–MS method for determination of azithromycin in human plasma by B. Barrett; V. Bořek-Dohalský; P. Fejt; S. Vaingátová; J. Huclová; B. Němec; I. Jelínek (pp. 210-217).
A validated, highly sensitive, and selective HPLC method with MS–MS detection has been developed for quantitative determination of azithromycin (AZI) in human Na2EDTA plasma. Roxithromycin (ROX) was used as internal standard. Human plasma containing AZI and internal standard was ultrafiltered through Centrifree Micropartition devices and the concentration of AZI was determined by isocratic HPLC–MS–MS. Multiple reaction monitoring mode (MRM) was used for MS–MS detection. The calibration plot was linear in the concentration range 2.55–551.43 ng mL−1. Inter-day and Intra-day precision and accuracy of the proposed method were characterized by R.S.D and percentage deviation, respectively; both were less than 8%. Limit of quantification was 2.55 ng mL−1. The proposed method was used to determine the pharmacokinetic profile of AZI (250-mg tablets).
Keywords: Azithromycin; HPLC; MS; Plasma; Pharmacokinetic study
Validated HPLC–MS–MS method for determination of azithromycin in human plasma by B. Barrett; V. Bořek-Dohalský; P. Fejt; S. Vaingátová; J. Huclová; B. Němec; I. Jelínek (pp. 210-217).
A validated, highly sensitive, and selective HPLC method with MS–MS detection has been developed for quantitative determination of azithromycin (AZI) in human Na2EDTA plasma. Roxithromycin (ROX) was used as internal standard. Human plasma containing AZI and internal standard was ultrafiltered through Centrifree Micropartition devices and the concentration of AZI was determined by isocratic HPLC–MS–MS. Multiple reaction monitoring mode (MRM) was used for MS–MS detection. The calibration plot was linear in the concentration range 2.55–551.43 ng mL−1. Inter-day and Intra-day precision and accuracy of the proposed method were characterized by R.S.D and percentage deviation, respectively; both were less than 8%. Limit of quantification was 2.55 ng mL−1. The proposed method was used to determine the pharmacokinetic profile of AZI (250-mg tablets).
Keywords: Azithromycin; HPLC; MS; Plasma; Pharmacokinetic study
Isolation and characterization of an anti-recombinant erythropoietin single-chain antibody fragment using a phage display antibody library by Jiebo Mi; Jin Yan; Zhenquan Guo; Meiping Zhao; Wenbao Chang (pp. 218-223).
The production of a large amount of specific antibodies against erythropoietin (EPO) is necessary for both clinical treatment and doping control. However, the weak immunogenicity of EPO and the side effects of excessive injection make the conventional immunological protocol rather inefficient and time-consuming. In this study, a single-chain antibody fragment of variable region (scFv) against recombinant human erythropoietin (rHuEPO) was produced after three rounds of panning a phage display antibody library. The selected scFv-B2 was expressed in soluble form in Escherichia coli DH5α F′ and purified by His-bond nickel affinity chromatography with a yield of about 1–2 mg of antibody in 1 L of the culture supernatant. The molecular weight of the scFv was estimated to be 29 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the affinity constant was found to be 1.0×108 L mol−1 based on a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). The potential ability of the scFvs for immunopurification of rHuEPO from related sample was demonstrated by using a double-antibody sandwich ELISA. The reported method is a very powerful tool to produce specific antibodies for rHuEPO detection demands.
Keywords: scFv; Erythropoietin; Phage display antibody library
Isolation and characterization of an anti-recombinant erythropoietin single-chain antibody fragment using a phage display antibody library by Jiebo Mi; Jin Yan; Zhenquan Guo; Meiping Zhao; Wenbao Chang (pp. 218-223).
The production of a large amount of specific antibodies against erythropoietin (EPO) is necessary for both clinical treatment and doping control. However, the weak immunogenicity of EPO and the side effects of excessive injection make the conventional immunological protocol rather inefficient and time-consuming. In this study, a single-chain antibody fragment of variable region (scFv) against recombinant human erythropoietin (rHuEPO) was produced after three rounds of panning a phage display antibody library. The selected scFv-B2 was expressed in soluble form in Escherichia coli DH5α F′ and purified by His-bond nickel affinity chromatography with a yield of about 1–2 mg of antibody in 1 L of the culture supernatant. The molecular weight of the scFv was estimated to be 29 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the affinity constant was found to be 1.0×108 L mol−1 based on a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). The potential ability of the scFvs for immunopurification of rHuEPO from related sample was demonstrated by using a double-antibody sandwich ELISA. The reported method is a very powerful tool to produce specific antibodies for rHuEPO detection demands.
Keywords: scFv; Erythropoietin; Phage display antibody library
Regional distributions of manganese, iron, copper, and zinc in the brains of 6-hydroxydopamine-induced parkinsonian rats by Tohru Tarohda; Yasushi Ishida; Keiichi Kawai; Masayoshi Yamamoto; Ryohei Amano (pp. 224-234).
Time courses of changes in manganese, iron, copper, and zinc concentrations were examined in regions of the brain of a 6-hydroxydopamine (6-OHDA)-induced rat model of Parkinson’s disease using inductively coupled plasma mass spectrometry (ICP-MS). The concentrations were simultaneously determined in brain section at the level of the substantia nigra 1, 3, 7, 10, 14, and 21 days after the 6-OHDA treatment and compared with those of control rats. The distributions of these elements were obtained for 18 regions of the sagittal section (1-mm thick). The ICP-MS results indicated that Mn, Fe, Cu, and Zn levels of the 6-OHDA-induced parkinsonian brain were observed to increase in all regions that lay along the dopaminergic pathway. In the substantia nigra, the increase in Mn level occurred rapidly from 3 to 7 days and preceded those in the other elements, reaching a plateau in the 6-OHDA brain. Iron and Zn levels increased gradually until 7 days and then increased rapidly from 7 to 10 days. The increase in the copper level was slightly delayed. In other regions, such as the globus pallidus, putamen, and amygdala, the levels of Mn, Fe, Cu, and Zn increased with time after 6-OHDA treatment, although the time courses of their changes were region-specific. These findings contribute to our understanding of the roles of Mn and Fe in the induction of neurological symptoms and progressive loss of dopaminergic neurons in the development of Parkinson’s disease. Manganese may hold the key to disturbing cellular Fe homeostasis and accelerating Fe levels, which play the most important role in the development of Parkinson’s disease.
Keywords: Manganese; Iron; Copper; Zinc; Brain; Parkinson’s disease
Regional distributions of manganese, iron, copper, and zinc in the brains of 6-hydroxydopamine-induced parkinsonian rats by Tohru Tarohda; Yasushi Ishida; Keiichi Kawai; Masayoshi Yamamoto; Ryohei Amano (pp. 224-234).
Time courses of changes in manganese, iron, copper, and zinc concentrations were examined in regions of the brain of a 6-hydroxydopamine (6-OHDA)-induced rat model of Parkinson’s disease using inductively coupled plasma mass spectrometry (ICP-MS). The concentrations were simultaneously determined in brain section at the level of the substantia nigra 1, 3, 7, 10, 14, and 21 days after the 6-OHDA treatment and compared with those of control rats. The distributions of these elements were obtained for 18 regions of the sagittal section (1-mm thick). The ICP-MS results indicated that Mn, Fe, Cu, and Zn levels of the 6-OHDA-induced parkinsonian brain were observed to increase in all regions that lay along the dopaminergic pathway. In the substantia nigra, the increase in Mn level occurred rapidly from 3 to 7 days and preceded those in the other elements, reaching a plateau in the 6-OHDA brain. Iron and Zn levels increased gradually until 7 days and then increased rapidly from 7 to 10 days. The increase in the copper level was slightly delayed. In other regions, such as the globus pallidus, putamen, and amygdala, the levels of Mn, Fe, Cu, and Zn increased with time after 6-OHDA treatment, although the time courses of their changes were region-specific. These findings contribute to our understanding of the roles of Mn and Fe in the induction of neurological symptoms and progressive loss of dopaminergic neurons in the development of Parkinson’s disease. Manganese may hold the key to disturbing cellular Fe homeostasis and accelerating Fe levels, which play the most important role in the development of Parkinson’s disease.
Keywords: Manganese; Iron; Copper; Zinc; Brain; Parkinson’s disease
Selenium metabolites in human urine after ingestion of selenite, L-selenomethionine, or DL-selenomethionine: a quantitative case study by HPLC/ICPMS by Doris Kuehnelt; Norbert Kienzl; Pedro Traar; Nam Hoang Le; Kevin A. Francesconi; Takafumi Ochi (pp. 235-246).
To obtain quantitative information on human metabolism of selenium, we have performed selenium speciation analysis by HPLC/ICPMS on samples of human urine from one volunteer over a 48-hour period after ingestion of selenium (1.0 mg) as sodium selenite, L-selenomethionine, or DL-selenomethionine. The three separate experiments were performed in duplicate. Normal background urine from the volunteer contained total selenium concentrations of 8–30 μg Se/L (n=22) but, depending on the chromatographic conditions, only about 30–70% could be quantified by HPLC/ICPMS. The major species in background urine were two selenosugars, namely methyl-2-acetamido-2-deoxy-1-seleno-β-D-galactopyranoside (selenosugar 1) and its deacylated analog methyl-2-amino-2-deoxy-1-seleno-β-D-galactopyranoside (selenosugar 3). Selenium was rapidly excreted after ingestion of the selenium compounds: the peak concentrations (∼250–400 μg Se/L, normalized concentrations) were recorded within 5–9 hours, and concentrations had returned to close to background levels within 48 hours, by which time 25–40% of the ingested selenium, depending on the species ingested, had been accounted for in the urine. In all experiments, the major metabolite was selenosugar 1, constituting either ∼80% of the total selenium excreted over the first 24 hours after ingestion of selenite or L-selenomethionine or ∼65% after ingestion of DL-selenomethionine. Selenite was not present at significant levels (<1 μg Se/L) in any of the samples; selenomethionine was present in only trace amounts (∼1 μg/L, equivalent to less than 0.5% of the total Se) following ingestion of L-selenomethionine, but it constituted about 20% of the excreted selenium (first 24 hours) after ingestion of DL-selenomethionine, presumably because the D form was not efficiently metabolized. Trimethylselenonium ion, a commonly reported urine metabolite, could not be detected (<1 μg/L) in the urine samples after ingestion of selenite or selenomethionine. Cytotoxicity studies on selenosugar 1 and its glucosamine isomer (selenosugar 2, methyl-2-acetamido-2-deoxy-1-seleno-β-D-glucosopyranoside) were performed with HepG2 cells derived from human hepatocarcinoma, and these showed that both compounds had low toxicity (about 1000-fold less toxic than sodium selenite). The results support earlier studies showing that selenosugar 1 is the major urinary metabolite after increased selenium intake, and they suggest that previously accepted pathways for human metabolism of selenium involving trimethylselenonium ion as the excretionary end product may need to be re-evaluated.
Keywords: Selenium metabolism; Selenosugar; HPLC; ICPMS; Urine
Selenium metabolites in human urine after ingestion of selenite, L-selenomethionine, or DL-selenomethionine: a quantitative case study by HPLC/ICPMS by Doris Kuehnelt; Norbert Kienzl; Pedro Traar; Nam Hoang Le; Kevin A. Francesconi; Takafumi Ochi (pp. 235-246).
To obtain quantitative information on human metabolism of selenium, we have performed selenium speciation analysis by HPLC/ICPMS on samples of human urine from one volunteer over a 48-hour period after ingestion of selenium (1.0 mg) as sodium selenite, L-selenomethionine, or DL-selenomethionine. The three separate experiments were performed in duplicate. Normal background urine from the volunteer contained total selenium concentrations of 8–30 μg Se/L (n=22) but, depending on the chromatographic conditions, only about 30–70% could be quantified by HPLC/ICPMS. The major species in background urine were two selenosugars, namely methyl-2-acetamido-2-deoxy-1-seleno-β-D-galactopyranoside (selenosugar 1) and its deacylated analog methyl-2-amino-2-deoxy-1-seleno-β-D-galactopyranoside (selenosugar 3). Selenium was rapidly excreted after ingestion of the selenium compounds: the peak concentrations (∼250–400 μg Se/L, normalized concentrations) were recorded within 5–9 hours, and concentrations had returned to close to background levels within 48 hours, by which time 25–40% of the ingested selenium, depending on the species ingested, had been accounted for in the urine. In all experiments, the major metabolite was selenosugar 1, constituting either ∼80% of the total selenium excreted over the first 24 hours after ingestion of selenite or L-selenomethionine or ∼65% after ingestion of DL-selenomethionine. Selenite was not present at significant levels (<1 μg Se/L) in any of the samples; selenomethionine was present in only trace amounts (∼1 μg/L, equivalent to less than 0.5% of the total Se) following ingestion of L-selenomethionine, but it constituted about 20% of the excreted selenium (first 24 hours) after ingestion of DL-selenomethionine, presumably because the D form was not efficiently metabolized. Trimethylselenonium ion, a commonly reported urine metabolite, could not be detected (<1 μg/L) in the urine samples after ingestion of selenite or selenomethionine. Cytotoxicity studies on selenosugar 1 and its glucosamine isomer (selenosugar 2, methyl-2-acetamido-2-deoxy-1-seleno-β-D-glucosopyranoside) were performed with HepG2 cells derived from human hepatocarcinoma, and these showed that both compounds had low toxicity (about 1000-fold less toxic than sodium selenite). The results support earlier studies showing that selenosugar 1 is the major urinary metabolite after increased selenium intake, and they suggest that previously accepted pathways for human metabolism of selenium involving trimethylselenonium ion as the excretionary end product may need to be re-evaluated.
Keywords: Selenium metabolism; Selenosugar; HPLC; ICPMS; Urine
LC-DAD-APCI-MS-based screening and analysis of the absorption and metabolite components in plasma from a rabbit administered an oral solution of danggui by Ya-Li Wang; Yi-Zeng Liang; Ben-Mei Chen; Yong-Kang He; Bo-Yan Li; Qian-Nan Hu (pp. 247-254).
A valid chromatographic fingerprint method using liquid chromatography-diode array detection-atmospheric pressure chemical ionization mass spectrometry in negative mode (LC-DAD-APCI-MS) is proposed for studying the absorption and metabolites of a traditional Chinese medicine (TCM) Angelica sinensis (danggui) in rabbit plasma, after the rabbit is administered with danggui oral solution (DOS). More than thirty-two common components were detected in both DOS and rabbit plasma, which shows that the components in the DOS were absorbed into the body of the rabbit. Of these, senkyunolide I, senkyunolide H, Z-6,7-epoxyligustilide, 3-butylidene-7-hydroxyphthalide, Z-ligustilide, Z-butylidenephthalide, Diels-Alder dimers of ligustilide, linolenic acid, linoleic acid and falcarindiol were tentatively identified from their MS, UV spectra and retention behavior by comparing the results with the published literature. At least ten components were found in rabbit plasma but not in DOS, indicating that these components must be metabolites of some of the components in the original extract. The results prove that the proposed method can be used to rapidly analyze multiple constituents in TCMs, and to screen for bioactive compounds by comparing and contrasting the chromatographic fingerprints of DOS and plasma samples.
Keywords: Chromatographic fingerprint; Screen and analyze; Absorption and metabolite components; Danggui oral solution; Rabbit plasma
LC-DAD-APCI-MS-based screening and analysis of the absorption and metabolite components in plasma from a rabbit administered an oral solution of danggui by Ya-Li Wang; Yi-Zeng Liang; Ben-Mei Chen; Yong-Kang He; Bo-Yan Li; Qian-Nan Hu (pp. 247-254).
A valid chromatographic fingerprint method using liquid chromatography-diode array detection-atmospheric pressure chemical ionization mass spectrometry in negative mode (LC-DAD-APCI-MS) is proposed for studying the absorption and metabolites of a traditional Chinese medicine (TCM) Angelica sinensis (danggui) in rabbit plasma, after the rabbit is administered with danggui oral solution (DOS). More than thirty-two common components were detected in both DOS and rabbit plasma, which shows that the components in the DOS were absorbed into the body of the rabbit. Of these, senkyunolide I, senkyunolide H, Z-6,7-epoxyligustilide, 3-butylidene-7-hydroxyphthalide, Z-ligustilide, Z-butylidenephthalide, Diels-Alder dimers of ligustilide, linolenic acid, linoleic acid and falcarindiol were tentatively identified from their MS, UV spectra and retention behavior by comparing the results with the published literature. At least ten components were found in rabbit plasma but not in DOS, indicating that these components must be metabolites of some of the components in the original extract. The results prove that the proposed method can be used to rapidly analyze multiple constituents in TCMs, and to screen for bioactive compounds by comparing and contrasting the chromatographic fingerprints of DOS and plasma samples.
Keywords: Chromatographic fingerprint; Screen and analyze; Absorption and metabolite components; Danggui oral solution; Rabbit plasma
Determination of trace proteins with pyronine Y and SDS by resonance light scattering by Suling Feng; Zihong Pan; Jing Fan (pp. 255-260).
A new resonance light scattering (RLS) probe for determining proteins is presented. The weak RLS of pyronine Y–SDS can be enhanced substantially by adding proteins in the presence of H2SO4, resulting in a strong and wide RLS band in the region 310–425 nm. The interaction of pyronine Y–SDS with proteins was studied on the basis of this behavior and a new quantitative method was developed for determining proteins. The enhanced RLS intensity is proportional to the concentration of proteins in the range 0.15–3.6 μg mL−1 for bovine serum albumin (BSA) and 0.06–4.8 μg mL−1 for human serum albumin (HSA), with detection limits of 21.0 and 12.0 ng mL−1, respectively. This method is characterized by high sensitivity, rapidity of reaction, and simplicity. Four synthetic samples were determined satisfactorily and recovery was 99.5–101.5%. Results for human serum and urine samples were in agreement with those obtained by the Bradford method, with relative standard deviations (RSD) of 1.5–3.1%.
Keywords: Resonance light scattering; Pyronine Y; SDS; Protein determination
Determination of trace proteins with pyronine Y and SDS by resonance light scattering by Suling Feng; Zihong Pan; Jing Fan (pp. 255-260).
A new resonance light scattering (RLS) probe for determining proteins is presented. The weak RLS of pyronine Y–SDS can be enhanced substantially by adding proteins in the presence of H2SO4, resulting in a strong and wide RLS band in the region 310–425 nm. The interaction of pyronine Y–SDS with proteins was studied on the basis of this behavior and a new quantitative method was developed for determining proteins. The enhanced RLS intensity is proportional to the concentration of proteins in the range 0.15–3.6 μg mL−1 for bovine serum albumin (BSA) and 0.06–4.8 μg mL−1 for human serum albumin (HSA), with detection limits of 21.0 and 12.0 ng mL−1, respectively. This method is characterized by high sensitivity, rapidity of reaction, and simplicity. Four synthetic samples were determined satisfactorily and recovery was 99.5–101.5%. Results for human serum and urine samples were in agreement with those obtained by the Bradford method, with relative standard deviations (RSD) of 1.5–3.1%.
Keywords: Resonance light scattering; Pyronine Y; SDS; Protein determination
Selective transport of copper(II) ions across a liquid membrane mediated by Piroxicam by Susan Sadeghi; Darush Mohammadzadeh; Jalal Shakhs Imampur (pp. 261-267).
Piroxicam was found to be a highly selective carrier for uphill transport of Cu2+ ions through a chloroform liquid membrane. The transport occurs via a counterflow of protons from the receiving phase to the source phase. The effects of several parameters on the transport of Cu2+ ions, such as the carrier concentration, pH of the source phase, composition of the receiving phase, and duration are described. A high transport efficiency (98±2%) was provided by the carrier for Cu2+ ions in a receiving phase of 0.01 mol l−1 sulfuric acid after 4 h. Different metal ion transport experiments showed that Cu2+ ions were selectively transported over other ions, such as Co2+, Ni2+, Cd2+, Pb2+, Zn2+, UO 2 2+ and ZrO 2 2+ . In the presence of fluoride ions (used as a suitable masking agent in the source phase), the interfering effects of UO 2 2+ and ZrO 2 2+ ions were eliminated. The applicability of the method was tested on a real sample, and the results obtained show that it is potentially useful for solvent extraction of copper.
Keywords: Liquid membrane; Copper transport; Piroxicam; Sulfuric acid; Real sample
Selective transport of copper(II) ions across a liquid membrane mediated by Piroxicam by Susan Sadeghi; Darush Mohammadzadeh; Jalal Shakhs Imampur (pp. 261-267).
Piroxicam was found to be a highly selective carrier for uphill transport of Cu2+ ions through a chloroform liquid membrane. The transport occurs via a counterflow of protons from the receiving phase to the source phase. The effects of several parameters on the transport of Cu2+ ions, such as the carrier concentration, pH of the source phase, composition of the receiving phase, and duration are described. A high transport efficiency (98±2%) was provided by the carrier for Cu2+ ions in a receiving phase of 0.01 mol l−1 sulfuric acid after 4 h. Different metal ion transport experiments showed that Cu2+ ions were selectively transported over other ions, such as Co2+, Ni2+, Cd2+, Pb2+, Zn2+, UO 2 2+ and ZrO 2 2+ . In the presence of fluoride ions (used as a suitable masking agent in the source phase), the interfering effects of UO 2 2+ and ZrO 2 2+ ions were eliminated. The applicability of the method was tested on a real sample, and the results obtained show that it is potentially useful for solvent extraction of copper.
Keywords: Liquid membrane; Copper transport; Piroxicam; Sulfuric acid; Real sample
A systematic approach to quantitation of ephedra alkaloids in natural health products by Joseph W. H. Lam; Graeme J. Gardner; Margaret McCooeye; Catharine A. Fraser; Ralph E. Sturgeon (pp. 268-281).
A method for accurate determination of ephedrine (E) alkaloids in natural health products (NHP) is described. The NIST dietary supplement standard reference materials (SRMs) were selected for these studies. These SRMs comprise ground Ma Huang herb (Ephedra sinica Stapf.), a spray dried extract of the former, and commercial formulations derived from gel caps and a protein drink. The efficiency of sonication-assisted extraction and Soxhlet extraction was studied using both ammonium formate and potassium phosphate in 3% methanol as extraction media. The efficiency of SPE clean-up of the extract deteriorated rapidly when increasing amounts of sample matrix or analyte were processed, because of limited cartridge capacity. Quantitation by the method of additions was required to ensure the highest accuracy using both LC–UV and ES–LC–MS–MS techniques. Whereas the LC–UV method is more convenient and precise, the results are more questionable than ES–LC–MS–MS, because species-specific detection is not possible.
Keywords: Ephedra; SRM; SPE clean-up; Standard addition calibration; LC–UV; LC–MS–MS
A systematic approach to quantitation of ephedra alkaloids in natural health products by Joseph W. H. Lam; Graeme J. Gardner; Margaret McCooeye; Catharine A. Fraser; Ralph E. Sturgeon (pp. 268-281).
A method for accurate determination of ephedrine (E) alkaloids in natural health products (NHP) is described. The NIST dietary supplement standard reference materials (SRMs) were selected for these studies. These SRMs comprise ground Ma Huang herb (Ephedra sinica Stapf.), a spray dried extract of the former, and commercial formulations derived from gel caps and a protein drink. The efficiency of sonication-assisted extraction and Soxhlet extraction was studied using both ammonium formate and potassium phosphate in 3% methanol as extraction media. The efficiency of SPE clean-up of the extract deteriorated rapidly when increasing amounts of sample matrix or analyte were processed, because of limited cartridge capacity. Quantitation by the method of additions was required to ensure the highest accuracy using both LC–UV and ES–LC–MS–MS techniques. Whereas the LC–UV method is more convenient and precise, the results are more questionable than ES–LC–MS–MS, because species-specific detection is not possible.
Keywords: Ephedra; SRM; SPE clean-up; Standard addition calibration; LC–UV; LC–MS–MS
Quantitative determination of Roundup Ready soybean (Glycine max) extracted from highly processed flour by Philippe Corbisier; Stefanie Trapmann; David Gancberg; Liesbeth Hannes; Pierre Van Iwaarden; Gilbert Berben; Heinz Schimmel; Hendrik Emons (pp. 282-290).
Roundup Ready soybean powder has been subjected to different amounts of DNA fragmentation to assess the accuracy of real-time PCR on processed food. Certified reference material (CRM) containing 10 g kg−1 of Roundup Ready soybean (ERM-BF410d) prepared by a dry-mixing processing method was exposed to water at two temperatures, using three different mixing devices, or to baking temperature (250°C) for 30 min. The amount of DNA extracted from the different samples was quantified by fluorimetry. The amount of fragmentation of the extracted DNA was characterised by gel and capillary electrophoresis and the percentage of genetically modified (GM) soybean was determined by a double quantitative real-time PCR method. Measurement of the event GTS 40-3-2 (RUR) was possible in all the treated materials, because small amplicons were amplified. Correct RUR percentages could be measured for intact powders with little or no DNA fragmentation. For samples with a high level of DNA degradation, however, the accuracy of the measurement was found to depend on the method used for DNA extraction. Genomic DNA isolated by use of silica resin resulted in statistically significant overestimation of the amount of GM.
Keywords: Glycine max; Genetically modified soybean (GMS); Polymerase chain reaction (PCR); Processed food; DNA degradation
Quantitative determination of Roundup Ready soybean (Glycine max) extracted from highly processed flour by Philippe Corbisier; Stefanie Trapmann; David Gancberg; Liesbeth Hannes; Pierre Van Iwaarden; Gilbert Berben; Heinz Schimmel; Hendrik Emons (pp. 282-290).
Roundup Ready soybean powder has been subjected to different amounts of DNA fragmentation to assess the accuracy of real-time PCR on processed food. Certified reference material (CRM) containing 10 g kg−1 of Roundup Ready soybean (ERM-BF410d) prepared by a dry-mixing processing method was exposed to water at two temperatures, using three different mixing devices, or to baking temperature (250°C) for 30 min. The amount of DNA extracted from the different samples was quantified by fluorimetry. The amount of fragmentation of the extracted DNA was characterised by gel and capillary electrophoresis and the percentage of genetically modified (GM) soybean was determined by a double quantitative real-time PCR method. Measurement of the event GTS 40-3-2 (RUR) was possible in all the treated materials, because small amplicons were amplified. Correct RUR percentages could be measured for intact powders with little or no DNA fragmentation. For samples with a high level of DNA degradation, however, the accuracy of the measurement was found to depend on the method used for DNA extraction. Genomic DNA isolated by use of silica resin resulted in statistically significant overestimation of the amount of GM.
Keywords: Glycine max; Genetically modified soybean (GMS); Polymerase chain reaction (PCR); Processed food; DNA degradation
Study of the conformation of γ-zeins in purified maize protein bodies by FTIR and NMR spectroscopy by Tatiana C. Bicudo; Lucimara A. Forato; Luiz A. R. Batista; Luiz A. Colnago (pp. 291-296).
The γ-zeins are a mixture of 16, 27, and 50-kDa polypeptides which are important in the formation and stabilization of protein bodies (PB). These organelles are used for deposition of zeins, the water-insoluble storage proteins in maize. The nature of the physical interaction between proteins in the assembly and stabilization of PB are fairly well known. It is suggested the repeated hexapeptide sequence (PPPVHL)8 in the N-terminus is responsible for aggregation of the γ-zeins on the PB surface. Despite this importance, there is little information about the native conformation of γ-zeins. In this work, we have analyzed the secondary structures of γ-zeins in purified protein bodies from two maize cultivars, in the solid state, by FTIR and NMR spectroscopy. The results revealed that γ-zeins in their physiological state are comprise similar proportions of α-helix and β-sheet, 33 and 31% as determined by FTIR. It was not possible to state if the polyproline II (PPII) conformation is present in the solid-state structure of γ-zeins, as has been demonstrated for the hexapeptide in solution. Because of the similarity of the solid-state NMR spectra of γ and α-zeins in the α carbon region we attributed their contributions to the β-sheet structures rather than to the PPII conformation or a mixture of these extended structures.
Keywords: γ-Zeins; Protein bodies; Secondary structure; FTIR; Solid-state NMR
Study of the conformation of γ-zeins in purified maize protein bodies by FTIR and NMR spectroscopy by Tatiana C. Bicudo; Lucimara A. Forato; Luiz A. R. Batista; Luiz A. Colnago (pp. 291-296).
The γ-zeins are a mixture of 16, 27, and 50-kDa polypeptides which are important in the formation and stabilization of protein bodies (PB). These organelles are used for deposition of zeins, the water-insoluble storage proteins in maize. The nature of the physical interaction between proteins in the assembly and stabilization of PB are fairly well known. It is suggested the repeated hexapeptide sequence (PPPVHL)8 in the N-terminus is responsible for aggregation of the γ-zeins on the PB surface. Despite this importance, there is little information about the native conformation of γ-zeins. In this work, we have analyzed the secondary structures of γ-zeins in purified protein bodies from two maize cultivars, in the solid state, by FTIR and NMR spectroscopy. The results revealed that γ-zeins in their physiological state are comprise similar proportions of α-helix and β-sheet, 33 and 31% as determined by FTIR. It was not possible to state if the polyproline II (PPII) conformation is present in the solid-state structure of γ-zeins, as has been demonstrated for the hexapeptide in solution. Because of the similarity of the solid-state NMR spectra of γ and α-zeins in the α carbon region we attributed their contributions to the β-sheet structures rather than to the PPII conformation or a mixture of these extended structures.
Keywords: γ-Zeins; Protein bodies; Secondary structure; FTIR; Solid-state NMR
Evaluating the mobility of toxic metals in untreated industrial wastewater sludge using a BCR sequential extraction procedure and a leaching test by T. G. Kazi; M. K. Jamali; G. H. Kazi; M. B. Arain; H. I. Afridi; A. Siddiqui (pp. 297-304).
The distribution and speciation of toxic metals in industrial wastewater sludge (IWS) was investigated. In this work, the modified BCR three-stage sequential extraction procedure was applied to the fractionation of Cr Pb Ni, and Cd in untreated industrial wastewater sludge from industrial sites in Hyderabad (Pakistan). The extracts were analyzed using electrothermal atomic absorption spectrometry. The procedure was evaluated using a certified reference material for soil mixed with sewage sludge BCR 483. The results from the partitioning study indicate that more easily mobilized forms (acid exchangeable) of Cd were dominant. The oxidizable fraction was dominant for all four toxic metals. Metal recovery was good, with <4% difference between the total metal recovered through the extractant steps and the total metal determined after microwave digestion. Lixiviation tests (DIN 38414-S4) were used to evaluate the leaching of toxic species from IWS, and it was observed that levels of leachable toxic metals were low compared to the amount of metal extracted in the exchangeable fraction of the BCR protocol.
Keywords: Industrial wastes; Toxic metals; BCR sequential extraction; Leaching procedure DIN 38414-S4; Electrothermal atomic absorption spectrometry
Evaluating the mobility of toxic metals in untreated industrial wastewater sludge using a BCR sequential extraction procedure and a leaching test by T. G. Kazi; M. K. Jamali; G. H. Kazi; M. B. Arain; H. I. Afridi; A. Siddiqui (pp. 297-304).
The distribution and speciation of toxic metals in industrial wastewater sludge (IWS) was investigated. In this work, the modified BCR three-stage sequential extraction procedure was applied to the fractionation of Cr Pb Ni, and Cd in untreated industrial wastewater sludge from industrial sites in Hyderabad (Pakistan). The extracts were analyzed using electrothermal atomic absorption spectrometry. The procedure was evaluated using a certified reference material for soil mixed with sewage sludge BCR 483. The results from the partitioning study indicate that more easily mobilized forms (acid exchangeable) of Cd were dominant. The oxidizable fraction was dominant for all four toxic metals. Metal recovery was good, with <4% difference between the total metal recovered through the extractant steps and the total metal determined after microwave digestion. Lixiviation tests (DIN 38414-S4) were used to evaluate the leaching of toxic species from IWS, and it was observed that levels of leachable toxic metals were low compared to the amount of metal extracted in the exchangeable fraction of the BCR protocol.
Keywords: Industrial wastes; Toxic metals; BCR sequential extraction; Leaching procedure DIN 38414-S4; Electrothermal atomic absorption spectrometry
An infrared spectroscopic method for quantitative analysis of fatty alcohols and fatty acid esters in machinery oils by Pekka Vähäoja; Jani Närhi; Toivo Kuokkanen; Outi Naatus; Jorma Jalonen; Sulo Lahdelma (pp. 305-311).
A new infrared spectroscopic method suitable for determining total fatty alcohol and fatty acid ester concentrations in industrial oils has been developed. Oil samples were diluted with toluene (1:3 w/w), the toxicity and volatility of which are relatively low compared with more commonly used IR solvents, like carbon tetrachloride or carbon disulfide. Mixture standards were prepared from dodecanol, tetradecanol, octadecanol, methyl stearate and methyl palmitate. Some analytical and statistical tests were performed on the developed method. The recoveries and the repeatability of the method proved to be sufficient for the quantitative determination of fatty alcohol and fatty acid ester additives in industrial oils. Reproducibility testing in another laboratory also produced satisfactory results. The developed method also proved to be relatively quick and simple. This method was developed to satisfy industry’s need to determine the concentrations of these oil additives, and it has already been applied successfully in machinery oil analysis.
Keywords: Fatty alcohols; Fatty acid esters; Infrared spectroscopy; Machinery oils; Quantitative analysis
An infrared spectroscopic method for quantitative analysis of fatty alcohols and fatty acid esters in machinery oils by Pekka Vähäoja; Jani Närhi; Toivo Kuokkanen; Outi Naatus; Jorma Jalonen; Sulo Lahdelma (pp. 305-311).
A new infrared spectroscopic method suitable for determining total fatty alcohol and fatty acid ester concentrations in industrial oils has been developed. Oil samples were diluted with toluene (1:3 w/w), the toxicity and volatility of which are relatively low compared with more commonly used IR solvents, like carbon tetrachloride or carbon disulfide. Mixture standards were prepared from dodecanol, tetradecanol, octadecanol, methyl stearate and methyl palmitate. Some analytical and statistical tests were performed on the developed method. The recoveries and the repeatability of the method proved to be sufficient for the quantitative determination of fatty alcohol and fatty acid ester additives in industrial oils. Reproducibility testing in another laboratory also produced satisfactory results. The developed method also proved to be relatively quick and simple. This method was developed to satisfy industry’s need to determine the concentrations of these oil additives, and it has already been applied successfully in machinery oil analysis.
Keywords: Fatty alcohols; Fatty acid esters; Infrared spectroscopy; Machinery oils; Quantitative analysis
Raman spectroscopic study of a post-medieval wall painting in need of conservation by Howell G. M. Edwards; Dennis W. Farwell; Christopher J. Brooke (pp. 312-321).
Raman spectroscopic studies of four specimens from an important angel wall painting in need of conservation work in a medieval church have provided some information about the pigments and pigment compositions which will influence possible future preservation and restoration strategies. Excitation of the Raman spectra at 1,064 nm in macroscopic mode and at 785 nm in microscopic mode revealed that the white pigment on the angel's wings was a mixture of barytes with calcite and lead white in minor composition. Although the specimens provided were not directly associated with coloured regions of the painting, yellow and blue microcrystals were found and they were identified as chrome yellow and lazurite, respectively. Red and brown particles were identified as cinnabar/vermilion and haematite. Several green particles were also found but could not be identified. The green and blue crystals could be related to neighbouring coloured regions of the artwork and the yellow colour could be identified as a background to the angel figure. Particles of carbon were found to be dispersed throughout the specimens and can be ascribed to soot from candles, heating stoves or oil lamps providing lighting in the church. No evidence for biological deterioration was found from the spectra. The unusual pigment palette is strongly suggestive of a later date of painting than was originally believed but there is a possibility that an earlier rendition exists underneath. Following a review of the spectroscopic data, a more extensive sampling protocol is recommended, from which some stratigraphic evidence could identify the underlying plaster and possible artwork.
Keywords: Raman spectroscopy; Wall painting; Conservation; Pigments
Raman spectroscopic study of a post-medieval wall painting in need of conservation by Howell G. M. Edwards; Dennis W. Farwell; Christopher J. Brooke (pp. 312-321).
Raman spectroscopic studies of four specimens from an important angel wall painting in need of conservation work in a medieval church have provided some information about the pigments and pigment compositions which will influence possible future preservation and restoration strategies. Excitation of the Raman spectra at 1,064 nm in macroscopic mode and at 785 nm in microscopic mode revealed that the white pigment on the angel's wings was a mixture of barytes with calcite and lead white in minor composition. Although the specimens provided were not directly associated with coloured regions of the painting, yellow and blue microcrystals were found and they were identified as chrome yellow and lazurite, respectively. Red and brown particles were identified as cinnabar/vermilion and haematite. Several green particles were also found but could not be identified. The green and blue crystals could be related to neighbouring coloured regions of the artwork and the yellow colour could be identified as a background to the angel figure. Particles of carbon were found to be dispersed throughout the specimens and can be ascribed to soot from candles, heating stoves or oil lamps providing lighting in the church. No evidence for biological deterioration was found from the spectra. The unusual pigment palette is strongly suggestive of a later date of painting than was originally believed but there is a possibility that an earlier rendition exists underneath. Following a review of the spectroscopic data, a more extensive sampling protocol is recommended, from which some stratigraphic evidence could identify the underlying plaster and possible artwork.
Keywords: Raman spectroscopy; Wall painting; Conservation; Pigments
Preparation and characterization of fused-silica capillary columns coated with m-carborane–siloxane copolymers for gas chromatography by Martina Petsch; Bernhard X. Mayer-Helm; Verena Söllner (pp. 322-326).
The carborane–siloxane copolymers Dexsil 300, a 34.5% bis(dimethylsilyl)-m-carborane–65.5% dimethylsiloxane copolymer, and Dexsil 400, a 24.9% bis(dimethylsilyl)-m-carborane–50.8% dimethyl, 24.3% methylphenylsiloxane copolymer, were coated on fused silica capillary columns and their gas chromatographic properties were evaluated. Their selectivity was evaluated using both Rohrschneider–McReynolds constants and triacylglycerol indices. The bis(dimethylsilyl)-m-carborane unit turned out to be equivalent to two dimethylsiloxy units and one half of a diphenylsiloxy unit. The m-carborane unit was found to cause a 15–25 K shift in the elution temperature between 120 and 360 °C. The working range was from 20 and 0 °C to 380 °C for Dexsil 300 and Dexsil 400, respectively. The column bleeding levels at 380 °C were below 20 and 15 pA for Dexsil 300 and Dexsil 400, respectively.
Keywords: m-Carborane–siloxane copolymers; Stationary phases; Gas chromatography; Selectivity; Equivalence model
Preparation and characterization of fused-silica capillary columns coated with m-carborane–siloxane copolymers for gas chromatography by Martina Petsch; Bernhard X. Mayer-Helm; Verena Söllner (pp. 322-326).
The carborane–siloxane copolymers Dexsil 300, a 34.5% bis(dimethylsilyl)-m-carborane–65.5% dimethylsiloxane copolymer, and Dexsil 400, a 24.9% bis(dimethylsilyl)-m-carborane–50.8% dimethyl, 24.3% methylphenylsiloxane copolymer, were coated on fused silica capillary columns and their gas chromatographic properties were evaluated. Their selectivity was evaluated using both Rohrschneider–McReynolds constants and triacylglycerol indices. The bis(dimethylsilyl)-m-carborane unit turned out to be equivalent to two dimethylsiloxy units and one half of a diphenylsiloxy unit. The m-carborane unit was found to cause a 15–25 K shift in the elution temperature between 120 and 360 °C. The working range was from 20 and 0 °C to 380 °C for Dexsil 300 and Dexsil 400, respectively. The column bleeding levels at 380 °C were below 20 and 15 pA for Dexsil 300 and Dexsil 400, respectively.
Keywords: m-Carborane–siloxane copolymers; Stationary phases; Gas chromatography; Selectivity; Equivalence model
Alkane effect in the Arizona liquid systems used in countercurrent chromatography by A. Berthod; M. Hassoun; M. J. Ruiz-Angel (pp. 327-340).
Countercurrent chromatography (CCC) is a separation technique that uses a biphasic liquid system; one liquid phase is the mobile phase, the other liquid phase is the stationary phase. Selection of the appropriate liquid system can be a problem in CCC, since it is necessary to select both the “column” and the mobile phase at the same time as the first is completely dependent on the second. A range of systems with various proportions of solvents were developed to ease this choice; 23 variations of the heptane/ethyl acetate/methanol/water biphasic liquid system were labeled A to Z. This range proved to be extremely useful and became the popular Arizona (AZ) liquid system. However, authors often replace the heptane with hexane. In this work, the chemical compositions of the upper phases and the lower phases of 55 Arizona systems made with various alkanes (pentane, hexane, heptane, isooctane and cyclohexane) were determined by gas chromatography and Karl Fischer titration. The test mixture separated consisted of five steroid compounds. The lower phases were found to have similar compositions when different alkanes were used, but the upper phases were found to change. Exchanging heptane for hexane or isooctane produced minimal changes in the CCC chromatogram, while changing the proportions of the solvents resulted in an exponential change in the retention volumes. The high density of cyclohexane made liquid stationary phase retention difficult. All Arizona systems equilibrated within 30 min, but were not stable: water slowly hydrolyzed the ethyl acetate (as shown by a continuous decrease in the pH of the lower aqueous phase), especially in the water-rich systems (early alphabet letters).
Keywords: Countercurrent chromatography; Biphasic liquid system; Phase composition; Elution-extrusion
Alkane effect in the Arizona liquid systems used in countercurrent chromatography by A. Berthod; M. Hassoun; M. J. Ruiz-Angel (pp. 327-340).
Countercurrent chromatography (CCC) is a separation technique that uses a biphasic liquid system; one liquid phase is the mobile phase, the other liquid phase is the stationary phase. Selection of the appropriate liquid system can be a problem in CCC, since it is necessary to select both the “column” and the mobile phase at the same time as the first is completely dependent on the second. A range of systems with various proportions of solvents were developed to ease this choice; 23 variations of the heptane/ethyl acetate/methanol/water biphasic liquid system were labeled A to Z. This range proved to be extremely useful and became the popular Arizona (AZ) liquid system. However, authors often replace the heptane with hexane. In this work, the chemical compositions of the upper phases and the lower phases of 55 Arizona systems made with various alkanes (pentane, hexane, heptane, isooctane and cyclohexane) were determined by gas chromatography and Karl Fischer titration. The test mixture separated consisted of five steroid compounds. The lower phases were found to have similar compositions when different alkanes were used, but the upper phases were found to change. Exchanging heptane for hexane or isooctane produced minimal changes in the CCC chromatogram, while changing the proportions of the solvents resulted in an exponential change in the retention volumes. The high density of cyclohexane made liquid stationary phase retention difficult. All Arizona systems equilibrated within 30 min, but were not stable: water slowly hydrolyzed the ethyl acetate (as shown by a continuous decrease in the pH of the lower aqueous phase), especially in the water-rich systems (early alphabet letters).
Keywords: Countercurrent chromatography; Biphasic liquid system; Phase composition; Elution-extrusion
Role of the activity coefficient in the dissemination of pH: comparison of primary (Harned cell) and secondary (glass electrode) measurements on phosphate buffer considering activity and concentration scales by Paola Fisicaro; Enzo Ferrara; Enrico Prenesti; Silvia Berto (pp. 341-348).
Despite recent efforts devoted to assessing both the theoretical rationale and the experimental strategy for assignment of primary pH values, these have not yet been accomplished satisfactorily. Traceability and comparability of pH values are achieved only within the constraints of internationally accepted conventions and predefined conditions that cannot account for all possible situations when pH is measured. Critical parameters to be defined are, in particular, the activity coefficients (γ i) of the ionic species involved in the equilibrium with the hydrogen ions in the solution, which are usually estimated with the approximation typical of the Debye–Hückel theoretical model. For this paper, primary (Harned cell) measurements (traceable to the SI system) of the pH of a phosphate buffer have been considered and the results have been compared with secondary (glass electrode) measurements obtained by considering either the activity (paH) or concentration (pcH) scale of the hydrogen ions. With conventional approaches based on measurements related to activity or concentration scale, discrepancies emerge which have been assigned to incomplete inferences of γ i arising from chemical features of the solution. It is shown that fitting and comparable paH and pcH results are attainable if evaluation of γ i is performed using better estimates of the ionic strength, according to an enhanced application of the Debye–Hückel theory.
Keywords: Primary pH measurement; Activity coefficients; Phosphate buffer; Ionic strength; Ionic medium
Role of the activity coefficient in the dissemination of pH: comparison of primary (Harned cell) and secondary (glass electrode) measurements on phosphate buffer considering activity and concentration scales by Paola Fisicaro; Enzo Ferrara; Enrico Prenesti; Silvia Berto (pp. 341-348).
Despite recent efforts devoted to assessing both the theoretical rationale and the experimental strategy for assignment of primary pH values, these have not yet been accomplished satisfactorily. Traceability and comparability of pH values are achieved only within the constraints of internationally accepted conventions and predefined conditions that cannot account for all possible situations when pH is measured. Critical parameters to be defined are, in particular, the activity coefficients (γ i) of the ionic species involved in the equilibrium with the hydrogen ions in the solution, which are usually estimated with the approximation typical of the Debye–Hückel theoretical model. For this paper, primary (Harned cell) measurements (traceable to the SI system) of the pH of a phosphate buffer have been considered and the results have been compared with secondary (glass electrode) measurements obtained by considering either the activity (paH) or concentration (pcH) scale of the hydrogen ions. With conventional approaches based on measurements related to activity or concentration scale, discrepancies emerge which have been assigned to incomplete inferences of γ i arising from chemical features of the solution. It is shown that fitting and comparable paH and pcH results are attainable if evaluation of γ i is performed using better estimates of the ionic strength, according to an enhanced application of the Debye–Hückel theory.
Keywords: Primary pH measurement; Activity coefficients; Phosphate buffer; Ionic strength; Ionic medium
Fluorescence ratiometric pH sensor prepared from covalently immobilized porphyrin and benzothioxanthene by Cheng-Gang Niu; Xiao-Qin Gui; Guang-Ming Zeng; Ai-Ling Guan; Pan-Feng Gao; Pin-Zhu Qin (pp. 349-357).
A fluorescence ratiometric sensor for pH determination is described in this paper. The sensor incorporated the pH-sensitive dye meso-5,10,15,20-tetra-(4-allyloxyphenyl)porphyrin (TAPP) as an indicator and a pH-insensitive dye N-(2-methacryloxyethyl)benzo[k,l]thioxanthene-3,4-dicarboximide (MBTD), a benzothioxanthene derivative, as a reference for fluorescence ratiometric measurement. To prevent leakage of the dyes, both were photocopolymerized with acrylamide, hydroxyethyl methacrylate, and triethylene glycol dimethacrylate on the silanized glass surface. The reproducibility and response time of the prepared sensor were sufficient. Most common coexisting inorganic ions and organic compounds did not interfere with pH sensing. In the acidic pH range from 1.5 to 5.0 the fluorescence intensity ratio of the two dyes varied linearly as a function of pH. The sensing membrane was found to have a lifetime of at least one month. The sensor was applied to the analysis of waste water and artificial samples.
Keywords: pH; Fluorescence ratiometric sensor; Covalent immobilization; Porphyrin; Benzothioxanthene
Fluorescence ratiometric pH sensor prepared from covalently immobilized porphyrin and benzothioxanthene by Cheng-Gang Niu; Xiao-Qin Gui; Guang-Ming Zeng; Ai-Ling Guan; Pan-Feng Gao; Pin-Zhu Qin (pp. 349-357).
A fluorescence ratiometric sensor for pH determination is described in this paper. The sensor incorporated the pH-sensitive dye meso-5,10,15,20-tetra-(4-allyloxyphenyl)porphyrin (TAPP) as an indicator and a pH-insensitive dye N-(2-methacryloxyethyl)benzo[k,l]thioxanthene-3,4-dicarboximide (MBTD), a benzothioxanthene derivative, as a reference for fluorescence ratiometric measurement. To prevent leakage of the dyes, both were photocopolymerized with acrylamide, hydroxyethyl methacrylate, and triethylene glycol dimethacrylate on the silanized glass surface. The reproducibility and response time of the prepared sensor were sufficient. Most common coexisting inorganic ions and organic compounds did not interfere with pH sensing. In the acidic pH range from 1.5 to 5.0 the fluorescence intensity ratio of the two dyes varied linearly as a function of pH. The sensing membrane was found to have a lifetime of at least one month. The sensor was applied to the analysis of waste water and artificial samples.
Keywords: pH; Fluorescence ratiometric sensor; Covalent immobilization; Porphyrin; Benzothioxanthene
Catalytic adsorptive stripping voltammetric determination of copper(II) on a carbon paste electrode by Ning Liu; Jun-Feng Song (pp. 358-364).
A catalytic adsorptive stripping voltammetric method for the determination of copper(II) on a carbon paste electrode (PCE) in an alizarin red S (ARS)-K2S2O8 system is proposed. In this method, copper(II) is effectively enriched by both the formation and adsorption of a copper(II)-ARS complex on the PCE, and is determined by catalytic stripping voltammetry. The catalytic enhancement of the cathodic stripping current of the Cu(II) in the complex results from a redox cycle consisting of electrochemical reduction of Cu(II) ion in the complex and subsequent chemical oxidation of the Cu(II) reduction product by persulfate, which reduces the contamination of the working electrode from Cu deposition and also improves analytical sensitivity. In Britton-Robinson buffer (pH 4.56±0.1) containing 3.6×10−5 mol L−1 ARS and 1.6×10−3 mol L−1 K2S2O8, with 180 s of accumulation at −0.2 V, the second-order derivative peak current of the catalytic stripping wave was proportional to the copper(II) concentration in the range of 8.0×10−10 to ∼3.0×10−8 mol L−1. The detection limit was 1.6×10−10 mol L−1. The proposed method was evaluated by analyzing copper in water and soil.
Keywords: Copper; Alizarin red S; Persulfate; Carbon paste electrode; Catalytic adsorptive stripping voltammetry
Catalytic adsorptive stripping voltammetric determination of copper(II) on a carbon paste electrode by Ning Liu; Jun-Feng Song (pp. 358-364).
A catalytic adsorptive stripping voltammetric method for the determination of copper(II) on a carbon paste electrode (PCE) in an alizarin red S (ARS)-K2S2O8 system is proposed. In this method, copper(II) is effectively enriched by both the formation and adsorption of a copper(II)-ARS complex on the PCE, and is determined by catalytic stripping voltammetry. The catalytic enhancement of the cathodic stripping current of the Cu(II) in the complex results from a redox cycle consisting of electrochemical reduction of Cu(II) ion in the complex and subsequent chemical oxidation of the Cu(II) reduction product by persulfate, which reduces the contamination of the working electrode from Cu deposition and also improves analytical sensitivity. In Britton-Robinson buffer (pH 4.56±0.1) containing 3.6×10−5 mol L−1 ARS and 1.6×10−3 mol L−1 K2S2O8, with 180 s of accumulation at −0.2 V, the second-order derivative peak current of the catalytic stripping wave was proportional to the copper(II) concentration in the range of 8.0×10−10 to ∼3.0×10−8 mol L−1. The detection limit was 1.6×10−10 mol L−1. The proposed method was evaluated by analyzing copper in water and soil.
Keywords: Copper; Alizarin red S; Persulfate; Carbon paste electrode; Catalytic adsorptive stripping voltammetry