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Analytical and Bioanalytical Chemistry (v.382, #5)

Building a professional career. Ethics—doing the right things by John Fetzer (pp. 1203-1205).

Mass spectrometry challenge by Juris Meija (pp. 1206-1206).

Solution to spectroscopy challenge 9 by Roberto Martínez-Alvarez; Antonio Herrera; Pedro Ramiro (pp. 1207-1208).

Fast fingerprinting by MALDI–TOF mass spectrometry of urinary sediment glycosphingolipids in Fabry disease by David Touboul; Sandrine Roy; Dominique P. Germain; Arlette Baillet; Françoise Brion; Patrice Prognon; Pierre Chaminade; Olivier Laprévote (pp. 1209-1216).

Fabry disease (FD) is an X-linked inborn error of glycosphingolipid (GSL) metabolism, caused by a deficiency of the lysosomal α-galactosidase A, which results in high levels in lysosomes and biological fluids of globotriaosylceramide (Gb₃) and digalactosylceramide (Ga₂), also known as galabiosylceramide. We report here a detailed study of the molecular species of GSLs in urinary samples obtained from hemizygous and heterozygous patients by use of matrix-assisted laser desorption ionisation and tandem mass spectrometry (MALDI–MS–MS). Twenty-two and fifteen molecular species were identified in the globotriaosylceramide and digalabiosylceramide series, respectively. The major sphingoid base was sphingosine (d18:1), and dihydrosphingosine (C18:0) and sphingadienine (d18:2) were also present. The molecular profiles obtained by MALDI–TOF-MS enabled us to show significant differences between GSLs composition for young, adult or atypic hemizygote and heterozygote patients. Thus, MALDI–TOF-MS and MS–MS proved a powerful tool for screening a population of patients with clinical signs suggestive of FD by direct and rapid GSL fingerprinting and identification, and for study of the biological processes occurring in glycosphingolipid accumulation.

Keywords: Mass spectrometry; MALDI–TOF; MALDI–MS–MS; Globotriaosylceramide; Digalactosylceramide; Fabry disease

Development of a competitive liposome-based lateral flow assay for the rapid detection of the allergenic peanut protein Ara h1 by Hsiao-Wei Wen; Wlodzimierz Borejsza-Wysocki; Thomas R. DeCory; Richard A. Durst (pp. 1217-1226).

A competitive lateral flow assay for detecting the major peanut allergen, Ara h1, has been developed. The detector reagents are Ara h1-tagged liposomes, and the capture reagents are anti-Ara h1 polyclonal antibodies. Two types of rabbit polyclonal antibodies were raised either against the entire Ara h1 molecules (anti-Ara h1 Ab) or against an immunodominant epitope on Ara h1 (anti-peptide Ab). All of them reacted specifically with Ara h1 in Western Blot against crude peanut proteins. Moreover, the anti-Ara h1 Ab was chosen for this assay development because of its highest immunoactivity to Ara h1-tagged liposomes in the lateral flow assay. The calculated limit of detection (LOD) of this assay is 0.45 μg mL⁻¹ of Ara h1 with a dynamic range between 0.1 and 10 μg mL⁻¹ of Ara h1 in buffer. Additionally, the visually determined detection range is from 1 to 10 μg mL⁻¹ of Ara h1 in buffer. Results using this assay can be obtained within 30 min without the need of sophisticated equipment or techniques; therefore, this lateral flow assay has the potential to be a cost-effective, fast, simple, and sensitive method for on-site screening of peanut allergens.

Keywords: Ara h1; Lateral flow assay; Liposomes; Peanuts; Peanut allergen

Separation of linear gramicidins using carbon dioxide-containing mobile phases by Kevin B. Thurbide; Jianmin Zhang (pp. 1227-1233).

Packed-column supercritical-fluid chromatography (pSFC) is presented as a novel method for separating and analyzing gramicidin samples. By use of methanol-modified carbon dioxide as a mobile phase the pentadecapeptides gramicidin A (gA), gramicidin B (gB), and gramicidin C (gC) are readily separated and eluted from a PRP-1 poly(styrene–divinylbenzene) column. Although optimum separation conditions are typically achieved near a column temperature of 40°C, a column pressure of 11 MPa, and 30% methanol modifier, pressure and modifier gradients around these values are also found to improve the overall separation time. Measurements indicate that the mobile phase solubility of gramicidin under these conditions is 5.0±0.4 μg mL⁻¹. Collection of individual peaks during chromatography achieved analytical-scale isolation of 2 μg refined gC from 20 μg injected gramicidin D. Further, supercritical-fluid extraction of 200 μg gramicidin D from a Chromosorb 102 support packed into the vessel produced 57 μg gA in 90% purity. The results establish that carbon dioxide-based mobile phases can be successfully used for the separation of individual gramicidin species.

Keywords: Supercritical; Carbon dioxide; Methanol; Modifier; Gramicidin; Separation

Amperometric determination of live Escherichia coli using antibody-coated paramagnetic beads by İsmail H. Boyacı; Zoraida P. Aguilar; Mahmud Hossain; H. Brian Halsall; Carl J. Seliskar; William R. Heineman (pp. 1234-1241).

Detecting and enumerating fecal coliforms, especially Escherichia coli, as indicators of fecal contamination, are essential for the quality control of supplied and recreational waters. We have developed a sensitive, inexpensive, and small-volume amperometric detection method for E. coli β-galactosidase by bead-based immunoassay. The technique uses biotin-labeled capture antibodies (Ab) immobilized on paramagnetic microbeads that have been functionalized with streptavidin (bead–Ab). The bead–Ab conjugate captures E. coli from solution. The captured E. coli is incubated in Luria Bertani (LB) broth medium with the added inducer isopropyl β-D-thiogalactopyranoside (IPTG). The induced β-galactosidase converts p-aminophenyl β-D-galactopyranoside (PAPG) into p-aminophenol (PAP), which is measured by amperometry using a gold rotating disc electrode. A good linear correlation (R²=0.989) was obtained between log cfu mL⁻¹ E. coli and the time necessary to product a specific concentration of PAP. Amperometric detection enabled determination of 2×10⁶ cfu mL⁻¹ E. coli within a 30 min incubation period, and the total analysis time was less than 1 h. It was also possible to determine as few as 20 cfu mL⁻¹ E. coli under optimized conditions within 6–7 h. This process may be easily adapted as an automated portable bioanalytical device for the rapid detection of live E. coli.

Keywords: Immunoassay; Amperometric detection; Paramagnetic beadsEscherichia coliβ-Galactosidase

Determination of atorvastatin and metabolites in human plasma with solid-phase extraction followed by LC–tandem MS by M. Hermann; H. Christensen; J. L. E. Reubsaet (pp. 1242-1249).

The aim of the present study was to develop a chromatographic method for the analysis of atorvastatin, o- and p-hydroxyatorvastatin (acid and lactone forms) in human plasma after administration of atorvastatin at the lowest registered dose (10 mg) in clinical studies. Sample preparation was performed by solid-phase extraction and was followed by separation of the analytes on an HPLC system with a linear gradient and a mobile phase consisting of acetonitrile, water and formic acid. Detection was achieved by tandem mass spectrometry operated in the electrospray positive ion mode. Validation of the method for the compounds for which reference compounds were available (acid forms of atorvastatin, o- and p-hydroxyatorvastatin) showed linearity within the concentration range (0.2–30 ng/ml for atorvastatin acid and p-hydroxyatorvastatin acid, and 0.5–30 ng/ml for o-hydroxyatorvastatin acid) (r²≥0.99, n=5 for all analytes). Accuracy and precision (evaluated at 0.5, 3 and 30 ng/ml for atorvastatin, p-hydroxyatorvastatin and 1, 3 and 30 ng/ml for o-hydroxyatorvastatin) were both satisfactory. The detection limit was 0.06 ng/ml for atorvastatin and p-hydroxyatorvastatin, and 0.15 ng/ml for o-hydroxyatorvastatin. The method has been successfully applied in a clinical study where atorvastatin, o- and p-hydroxyatorvastatin (both acid and lactone forms) could be detected in a 24-h sampling interval after administration of the lowest registered dose of atorvastatin (10 mg) for one week.

Keywords: Atorvastatin; Metabolites; Solid-phase extraction; Interconversion

Chemiluminescence immunoassay for chloramphenicol by Si Lin; Shi-quan Han; Yi-bing Liu; Wen-ge Xu; Guo-ying Guan (pp. 1250-1255).

In recent years, various chemiluminescent clinical immunoassay kits have been widely applied to the detection of hormones. However, a kit for chloramphenicol (CAP) is often absent from most commercial product lists, even though it is important to control the levels of CAP residues in foodstuffs too. Therefore, we describe a simple, solid-phase chemiluminescence immunoassay (CLIA) for the measurement of CAP in foodstuffs. A rabbit anti-CAP IgG is passively adsorbed onto the walls of polypropylene plates. The labeled antigen is horseradish peroxidase (HRP) conjugate of CAP. Luminol solution is used as the substrate of HRP. The light yield is inversely proportional to the concentration of CAP. The method has a similar sensitivity (0.05 ng/ml), specificity, precision, and accuracy to a conventional enzyme immunoassay (EIA). The intra-assay and inter-assay CVs of ten samples were <8% and <20%, respectively, and the analytical recovery of the method was 87–100%. The experimental correlation coefficient of dilution was found to be 0.999 using milk supernatant as buffer. The detection limit for the method was 0.1–10 ng/ml, and it displayed good linearity.

Keywords: Chloramphenicol (CAP); Chemiluminescence immunoassay (CLIA); Horseradish peroxidase (HRP)

Effects of diclofenac on EPC liposome membrane properties by Helena Ferreira; Marlene Lúcio; José L. F. C. Lima; Carla Matos; Salette Reis (pp. 1256-1264).

In this work the interaction of a non-steroidal anti-inflammatory drug (NSAID), diclofenac, with egg yolk phosphatidylcoline (EPC) liposomes, used as cell-membrane models, was quantified by determination of the partition coefficient. The liposome/aqueous phase partition coefficient was determined by derivative spectrophotometry, fluorescence quenching, and measurement of zeta-potential. Theoretical models based on simple partition of the diclofenac between two different media, were used to fit the experimental data, enabling the determination of K_p. The three techniques used yielded similar results. The effects of the interaction on the membrane’s characteristics were further evaluated, either by studying membrane potential changes or by effects on membrane fluidity. The liposome membrane potential and the size and size-homogeneity of liposomes were measured by light scattering. The effects of diclofenac on the internal viscosity or fluidity of the membrane were determined by use of spectroscopic probes—a series of n-(9-anthroyloxy) fatty acids in which the carboxyl terminal group is located at the interfacial region of the membrane and the fluorescent anthracene group is attached at different positions along the fatty acid chain. The location of the diclofenac on the membrane was also evaluated, by fluorescence quenching using the same series of fluorescent probes. Because the fluorescent anthracene group is attached at different positions along the fatty acid chain, it is possible to label at a graded series of depths in the bilayer. The interactions between the drug and the probe are a means of predicting the location of the drug on the membrane.

Keywords: Diclofenac; Liposomes; Derivative spectrophotometry; Fluorescence quenching; Anisotropy; Zeta-potential

Pre-column derivatization of rofecoxib for determination in serum by HPLC by M. Amini; M. PirAli Hamedani; M. Vosooghi; M. Nabavi; A. Shafiee (pp. 1265-1268).

An HPLC method for determination of rofecoxib in human serum is presented. The method is based on pre-column derivatization of analyte to a phenanthrene derivative of the drug. Rofecoxib and the internal standard were extracted from serum using liquid–liquid extraction. Upon exposure to UV light, the drug was found to undergo a photocyclization reaction, giving a species with high absorbance. Validation of the method has been studied in the concentration range 10–500 ng ml⁻¹.

Keywords: Rofecoxib; HPLC; Serum; UV detection

Experimental design-based development and single laboratory validation of a capillary zone electrophoresis method for the determination of the artificial sweetener sucralose in food matrices by Josephine McCourt; Joerg Stroka; Elke Anklam (pp. 1269-1278).

A capillary zone electrophoresis (CZE) method, optimised chemometrically, underwent a complete in-house validation protocol for the qualification and quantification of sucralose in various foodstuffs. Separation from matrix components was obtained in a dinitrobenzoic acid (3 mM)/sodium hydroxide (20 mM) background electrolyte with a pH of 12.1, a potential of 0.11 kV cm⁻¹ and a temperature of 22 °C. Detection was achieved at 238 nm by indirect UV. Screening, optimisation and robustness testing were all carried out with the aid of experimental design. Using standard addition calibration, the CZE method has been applied to still, carbonated and alcoholic beverages, yoghurts and hard-boiled candy. The method allows the detection of sucralose at >30 mg kg⁻¹, with a linearity range of 50–500 mg kg⁻¹, making it suitable for implementation of the recently amended “Sweeteners for use in foodstuffs” Directive (European Parliament and Council (2003) Off J L237:3–12), which set maximum usable doses of sucralose for many foodstuffs, most ranging from 200 mg kg⁻¹ to 450 mg kg⁻¹.

Keywords: Capillary electrophoresis; Sucralose; Experimental design; Validation; Food

Characterisation of selenium compounds in rye seedling biomass using ⁷⁵Se-labelling/SDS-PAGE separation/γ-scintillation counting, and HPLC-ICP-MS analysis of a range of enzymatic digests by Malgorzata A. Bryszewska; Wojciech Ambroziak; Juliusz Rudzinski; D. John. Lewis (pp. 1279-1287).

In the present study, selenium-enriched plant biomass was investigated to evaluate the ability of rye seedlings to take up, and assimilate, inorganic selenium. Two different analytical approaches were used. Electrophoretic separation (SDS-PAGE) of proteins extracted from ⁷⁵Se-labelled biomass was used to investigate the biotransformation of selenite into organic forms of the element. Ion-pair chromatography coupled with ICP-MS detection was chosen for the analysis of selenium species, enzymatically extracted from the plant biomass. The results of three enzymatic hydrolysis procedures and three sequential enzymatic extractions procedures are compared. The most effective single extraction was proteolysis (using protease type XIV), giving an overall extraction efficiency of 48%. However, for combinations of enzymes, the most effective was cellulase (Trichoderma viride) followed by sequential extraction of the solid pellet using protease type XIV, giving an extraction efficiency of 70%. The complementary data from the electrophoretic fractionation of proteins, and the HPLC separation of Se-species in the proteolytic digests, reveal the existence of large number of selenium-containing compounds in the rye seedling plant biomass. The results showed the complete biotransformation of inorganic selenium into organic forms during germination of the rye seedlings. HPLC-ICP-MS analysis of extracts from the plant biomass did not show the presence of selenate or selenite. At the time of this study, the lack of suitable organic-MS facilities meant that it was not possible to characterise them fully. However, the data does show that a combination of different enzymes, rather than just the commonly-used protease, should be considered when developing an extraction strategy for selenium in different food types to those already reported in the literature.

Keywords: Selenium; Speciation; Selenoproteins; Plant biomass; Selenomethionine; SDS-PAGE; Scintillation counting; HPLC-ICP-MS

Determination of ochratoxin A in Portuguese rice samples by high performance liquid chromatography with fluorescence detection by A. Pena; F. Cerejo; C. Lino; I. Silveira (pp. 1288-1293).

A sensitive and accurate analytical method for the determination of ochratoxin A (OTA) in rice, based on extraction with phosphate-buffered saline/methanol, an immunoaffinity column (IAC) for clean-up, and high performance liquid chromatography with fluorescence detection (HPLC-FD), is described. The limit of quantification of the proposed method was 0.05 μg kg⁻¹. Recovery of OTA from rice samples spiked at 0.05 μg kg⁻¹ was 92%, with a within-day RSD of 5.4%. The proposed method was applied to 42 rice samples from Portugal and the presence of OTA was found in six samples at concentrations ranging from 0.09 to 3.52 μg kg⁻¹. The identification of OTA was confirmed by methyl ester derivatization and then HPLC analysis. The daily intake of OTA by the Portuguese population was also estimated.

Keywords: Food analysis; Mycotoxins; Ochratoxin A; Rice; HPLC; Fluorescence detection

Investigation of a cylindrical chemosorptive denuder for sampling and phase separation of toluene diisocyanate aerosols by Yvonne Nordqvist; Ulrika Nilsson; Anders Colmsjö (pp. 1294-1299).

A cylindrical chemosorptive denuder in series with a glass fibre filter has been evaluated for sampling toluene diisocyanate (TDI) aerosols. The sampler is designed for measuring personal exposure to diisocyanates. Several denuder coatings and derivatising reagents were investigated. Dimethylpolysiloxane (SE-30) and 5% phenyl dimethylpolysiloxane (SE-54) with either dibutylamine (DBA) or dipentylamine (DPeA) as derivatising reagents yielded the lowest vapour breakthrough (the amount (%) of the vapour that passes through the denuder), close to values predicted by theory. Immobilisation of the SE-30 denuder coating by in-situ cross-linking yielded comparable results. With an SE-30/DBA-coated denuder operating within an airflow range of 100–500 mL min⁻¹, the phase separation was shown to be consistent with theoretical predictions derived by use of the Gormley–Kennedy equation. This provides a means of calculating the vapour breakthrough and correcting experimentally obtained values with regard to vapour–particulate phase distribution, suggesting that the denuder can provide accurate phase-distribution measurements. The SE-30/DBA denuder can be used over a concentration range spanning nearly six orders of magnitude. Its capacity is sufficient to perform 15-min exposure measurements of a TDI aerosol with air concentrations as high as 1,700 μg m⁻³, 40 times higher than the Swedish occupational exposure limit (OEL). At the other end of the range, the estimated limit of detection (LOD) was less than 2 ng m⁻³ for both the vapour and the aerosol phases when LC–ESI–MS–MS was used for chemical analysis.

Keywords: Cylindrical denuder; Toluene diisocyanate; TDI; Aerosol sampling; Chemosorptive sampling

Using organic nanoparticle fluorescence to determine nitrite in water by Lun Wang; Ling Dong; Gui-Rong Bian; Le-Yu Wang; Ting-Ting Xia; Hong-Qi Chen (pp. 1300-1303).

Organic fluorescence nanoparticles (1-aminopyrene nanoparticles) were prepared under ultrasonic radiation and used to determine nitrite. Such nanoparticles have a broad, continuous excitation spectrum, but they are also photochemically stable and water-soluble. Nitrite determination was based upon nitrite quenching the fluorescence of the nanoparticles due to a simple diazotization reaction; a simple and specific method. Under optimal conditions, the linear range of the calibration curve was 20–1400 ng ml⁻¹, with a correlation coefficient of 0.9987 for nitrite. The detection limit was 3 ng ml⁻¹. The method was applied to various water samples from several sources. Quantitative nitrite recoveries and satisfactory results were achieved.

Keywords: Fluorescence probe; Nitrite; Organic nanopartocles; Diazotization

Predicting gas chromatographic retention times of 209 polybrominated diphenyls (PBBs) for different temperature programs by H. X. Zhao; Q. Zhang; X. Y. Xue; X. M. Liang; A. Kettrup (pp. 1304-1310).

A method has been developed to predict the retention times of 209 individual polybrominated diphenyl congeners for different temperature programs. The retention equations lnk‘=A+B/T of five PBBs in gas chromatography (GC) were used to evaluate the properties of the regression coefficients A and B, which are widely accepted as being highly reliable chromatographic retentions. The quantitative relationships between the A and B values of PCBs and those of PBBs were found. The regression equations derived have coefficients of determination greater than 0.999. The A, B values of any PBB can be predicted by using the A, B values of the PCB according to these relationships. Using these predicted A and B values, the retention times of all PBBs can be predicted. This is an important advance in the identification of PBBs because at present there are only a few PBB standards available.

Keywords: Chromatographic retention; Prediction; PBBs; PCBs quantitative relationship

Integration of metrological principles in a proficiency-testing scheme in the field of water analysis by Andreja Drolc; Magda Cotman; Milenko Roš (pp. 1311-1319).

Measurement traceability is universally recognised as one of the basic prerequisites for comparability of results obtained in different laboratories and is a basic aspect of metrological sciences such as analytical chemistry. This requirement is underscored by the increasing adoption of standards and measurement quality systems, such as laboratory accreditation against ISO/IEC 17025. Testing laboratories ensure traceability of their measurement results by using appropriate reference standards for calibration of instruments and control of measurement processes. For routine work in the field of water analysis, these standards are usually commercial solutions or in-house solutions prepared from “pure” products. Therefore, laboratories should demonstrate that their use of reference standards is appropriate and sufficient, which can be done by participation in an appropriate proficiency-testing scheme. The paper reports how measurement traceability of results from field laboratories (nitrite nitrogen, nitrate nitrogen, chloride and sulphate; all in water) can be demonstrated by participation in a proficiency-testing scheme based on reference values.

Keywords: Wastewater analysis; Traceability; Proficiency testing; Reference value

Optimization of an analytical procedure for the determination of banned azo dyes in leather by Lars-Henric Ahlström; Erland Björklund; Lennart Mathiasson (pp. 1320-1327).

The possibility of improving an existing method, based on supercritical-fluid extraction (SFE) and microwave-assisted extraction (MAE), for the determination of banned azo dyes in leather has been studied. Thus, optimization of experimental conditions in different steps (degreasing, reduction, and extraction) of the analytical procedure was performed. The influence of different variables (reaction time, temperature, and concentration of reducing agent) on the reduction process was evaluated by use of a factorial design. It was found that the concentration of the reducing agent and the interaction between time and temperature were the most influential variables. Consequently, by applying a higher temperature, the reaction time could be halved. The use of acidified water as extraction solvent in MAE was also investigated. Usually 1 mol L⁻¹ HCl was superior to methanol and buffer in terms of extraction efficiency. In conclusion, the present method gave significantly higher recoveries in comparison with the original method. All dyes were determined indirectly by measuring their corresponding harmful amines, formed after reduction by use of sodium dithionite.

Keywords: Azo dyes; Aromatic amines; Analytical procedure; Extraction; Factorial design; Leather

Redox-iodometry: a new potentiometric method by Waldemar Gottardi; Jörg Pfleiderer (pp. 1328-1338).

A new iodometric method for quantifying aqueous solutions of iodide-oxidizing and iodine-reducing substances, as well as plain iodine/iodide solutions, is presented. It is based on the redox potential of said solutions after reaction with iodide (or iodine) of known initial concentration. Calibration of the system and calculations of unknown concentrations was performed on the basis of developed algorithms and simple GWBASIC-programs. The method is distinguished by a short analysis time (2–3 min) and a simple instrumentation consisting of pH/mV meter, platinum and reference electrodes. In general the feasible concentration range encompasses 0.1 to 10⁻⁶ mol/L, although it goes down to 10⁻⁸ mol/L (0.001 mg Cl₂/L) for oxidants like active chlorine compounds. The calculated imprecision and inaccuracy of the method were found to be 0.4–0.9% and 0.3–0.8%, respectively, resulting in a total error of 0.5–1.2%. Based on the experiments, average imprecisions of 1.0–1.5% at c(Ox)>10⁻⁵ M, 1.5–3% at 10⁻⁵ to 10⁻⁷ M, and 4–7% at <10⁻⁷ M were found. Redox-iodometry is a simple, precise, and time-saving substitute for the more laborious and expensive iodometric titration method, which, like other well-established colorimetric procedures, is clearly outbalanced at low concentrations; this underlines the practical importance of redox-iodometry.

Keywords: Redox potential; Oxidation capacity; Iodometric titration; Colorimetric methods

The use of indium(III) as a complexing counter-ion to enable the separation of chloride, bromide, and iodide using isotachophoresis by Jeff E. Prest; Peter R. Fielden (pp. 1339-1342).

A new method has been devised to enable the determination of halide anions by isotachophoresis. This method uses an electrolyte system that employs indium(III) as a counter-ion to manipulate the effective mobilities of sample species by means of complexation reactions. This new procedure successfully enabled the simultaneous determination of the halide ions chloride, bromide, and iodide when a 12 mmol L⁻¹ nitrate-based leading electrolyte containing 3.5 mmol L⁻¹ indium(III) at pH 3.0 was used.

Keywords: Isotachophoresis; Electrophoresis; Halide separation

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Analytical and Bioanalytical Chemistry (v.382, #5)