Robert Q. Thompson

Comparison of methods for following alkaline phosphatase catalysis: Spectrophotometric versus amperometric detection

An amperometric method for alkaline phosphatase is described and compared to the most widely used spectrophotometric method. Catalytic hydrogenation of 4-nitrophenylphosphate (the substrate in the spectrophotometric method) gives 4-aminophenylphosphate (the substrate in the amperometric method). The latter substrate has the formula C6H6NO4PNa2.5H2O and a Mr of 323. The Michaelis constant for 4-aminophenylphosphate in 0.10 M, pH 9.0. Tris buffer is 56 microM, while it is 82 microM for 4-nitrophenyl phosphate. The amperometric method has a detection limit of 7 nM for the product of the enzyme reaction, which is almost 20 times better than the spectrophotometric method. Similarly, with a 15-min reaction at room temperature and in a reaction volume of 1.1 ml, 0.05 microgram/l alkaline phosphatase can be detected by electrochemistry, almost an order of magnitude better than by absorption spectrophotometry. Amperometric detection is ideally suited for small-volume and trace immunoassay.

Zeptomole detection limit for alkaline phosphatase using 4-aminophenylphosphate, amperometric detection, and an optimal buffer system

Abstract Trace detection of bovine alkaline phosphatase was studied using the substrate, 4-aminophenylphosphate, and amperometric detection of the product, 4-aminophenol. Six aminoethanol buffers were compared with respect to their abilities to stabilize the product and to promote enzyme activity. In ethanolamine and 2-(methylamino)ethanol buffers, progress curves were linear from zero time to at least 60 min, while progress curves in the other buffers became non-linear in a short time due to product decomposition. The highest reaction rate at pH 10.0 was found in the 2-(methylamino)ethanol buffer, while the highest pseudo-rate contant, V max / V m , was found in tris(hydroxymethyl-aminomethane. Higher buffer concentration and the addition of magnesium ion enhanced enzyme activity, while the addition of zinc ion decreased enzyme activity. In 1.0 F, pH 10.0, 2-(methylamino)ethanol buffer and in the presence of 1 mg ml −1 MgCl 2 , the detection limit for aqueous mouse IgG-alkaline phosphatase conjugate was 25 amol ml −1 or 500 zeptomoles (10 −21 mol). The reaction time was 60 min. Under similar condition, the detection limit for IgG-alkaline phosphate adsorbed onto a microtiter plate was 4 attomoles.

AQUEOUS RECOVERY FROM COTTON SWABS OF ORGANIC EXPLOSIVES RESIDUE FOLLOWED BY SOLID PHASE EXTRACTION

A common procedure for processing cotton swabs containing organic explosives residue involves soaking the cotton in acetone or other organic solvent to extract the explosives, followed by direct analysis of the resulting sample solution using chromatography—mass spectrometry (LC- or GC-MS). A water-based procedure was developed to solve problems arising from co-extraction of sample matrix. Common nitro-organic explosives were extracted from cotton into water; the explosives were isolated by solid phase extraction, using a poly-N-vinylpyrrolidone-divinylbenzene sorbent; samples were screened by LC-UV; and the presence of explosives was confirmed by LC- or GC-MS and fast GC-TEA (EGIS). Explosives residue samples were generated by mixing standards in motor oil on aluminum foil, by detonating four different bombs (C-4, a dynamite, a binary explosive, and TNT) hidden inside suitcases filled with clothing, and by handling a plastic explosive (Semtex H). Ninety-six paired samples were processed by the two procedures (acetone-based and water-based). The water extraction/SPE process was just as effective in recovering organic explosives from cotton swabs, and it better rejected the sample matrix, giving much greater selectivity with all samples except clothing. Water-based samples were screened with high accuracy by LC-UV, and the LC-UV and LC-MS semi-quantitative results were highly correlated.

Novel separation for the determination of cadmium by isotope dilution ICP-MS in samples containing high concentrations of molybdenum and tin

A four-step matrix reduction procedure, beginning with thiourea-based solid phase extraction, was developed for the determination of cadmium in difficult sample matrixes by inductively coupled plasma mass spectrometry. The separation scheme reduced the concentrations of tin and molybdenum, significant spectral interference sources for cadmium, from mg kg−1 levels to μg kg−1 and sub-μg kg−1 levels respectively, making possible interference-free measurement of cadmium and facilitating isotope dilution quantification. Cadmium recovery through the matrix reduction procedure was 78%. The effectiveness of the method was shown by application to NIST SRMs 1568a Rice Flour and 2703 Marine Sediment.

Comparison of immobilized enzyme reactors for flow-injection systems

Abstract Straight, coiled, beaded, and packed-bed reactors containing immobilized glucose oxidase or l -(+)-lactate dehydrogenase were compared in terms of reaction rate, sensitivity, sample throughput, and sample dispersion. The enzymes were covalently attached to the inside walls of 5.0-cm long, 1.12-mm i.d. nylon tubes, and the resulting reactors were tested in a flow-injection system. The beaded enzymatically-active reactors (BEARs) were filled with solid glass beads of 0.5-mm or 1.0-mm diameter. Reactors with the larger beads had 2–4 times the activity, twice the sensitivity, and better throughput than the open reactors; they also minimized the physical and chemical contributions to dispersion. Packed-bed reactors were superior in the lactate determination, but the beaded reactors were better for the determination of glucose. With BEARs containing 1.0-mm beads, glucose was determined in the range 10–800 μM with a conversion efficiency of 0.056 mol of product per mole of substrate; for lactate, the range was 8–64 μM with a conversion efficiency of 0.13 mol mol−1.

Determination of trace level cadmium in SRM 3280 Multivitamin/Multielement Tablets via isotope dilution inductively coupled plasma mass spectrometry

Cadmium was quantified at 80.15±0.86 ng/g (mean±95% expanded uncertainty) in NIST SRM 3280 Multivitamin/Multielement Tablets, using isotope dilution mass spectrometry. The method described utilized various precipitation and solid-phase extraction separation methodologies to isolate Cd from Sn and Mo, present respectively, at 11.1±0.9 mg/kg and 70.7±4.5 mg/kg in the tablet matrix. This allowed for measurement of (111)Cd/(113)Cd and (111)Cd/(114)Cd isotope ratios using both quadrupole collision cell technology inductively coupled plasma mass spectrometry (Q-CCT-ICP-MS) and sector field (SF)-ICP-MS equipped with a desolvating nebulizer system to mitigate the MoO(+) and MoOH(+) molecular ion interferences that typically affect the envelope of Cd isotopes.

Construction and evaluation of a regenerable fluoroimmunochemical-based fibre optic biosensor

A microscale fibre optic biosensor that is capable of in situ regeneration is described and characterized. By combining recently developed fibre optic sensing technology with a capillary column reagent delivery system, it is possible to perform a variety of bench-top affinity assay procedures both repetitively and remotely. The configuration of the sensing chamber at the terminus of the fibre is an important design feature. The construction and operation of the sensor is described and the results of evaluations of the sensor using an antibody–antigen system are presented. Affinity assay steps such as the delivery of solid phase affinity reagents, secondary reagents and rinse solutions are demonstrated. Sampling is accomplished by mild aspiration. Relative standard deviations (RSDs) for these steps are all less than 10%. The capability of selectively measuring fluorescently labelled anti-rabbit immunoglobulin G (lgG), in the presence of a similar protein, by utilizing its immunospecific interaction with rabbit lgG immobilized on silica beads is demonstrated, and exhibits an RSD of 6.2%. A near linear calibration graph is presented over a concentration range of between 0.011 (approximately the limit of detection) and 0.11 mg ml–1.

Isolation of individual capsaicinoids from a mixture and their characterization by 13C NMR spectrometry

Individual compounds were isolated from a laboratory mixture of capsaicinoids by a multi-stage approach. First, the capsaicinoids were fractionated into capsaicins and non-capsaicins by argentation solid phase extraction (SPE) on a silver-charged propyl sulfonate resin. Second, compounds in each fraction were isolated by semi-preparative liquid chromatography on a C(30) phase in aqueous methanol. Third, the individual components of the original mixture were concentrated by reversed phased (C(18)) SPE. The structure of each purified compound was confirmed by (13)C NMR spectrometry and spectral comparison to known standards, purchased or synthesized locally. The chemical shifts of 15 capsaicinoid standards were measured on a 600MHz instrument, and their assignments to particular carbons were made by reference to Distortionless Enhancement by Polarization Transfer (DEPT) NMR experiments and NMR spectral prediction software.

Solid-Phase Determination of Glucose in Whole Blood

Abstract A solid-phase determination of glucose in whole blood has been developed that employs an immobilized substrate and aqueous enzymes. A phenol, 3-hydroxyphenylacetic acid, was covalently bonded to micron-size porous glass beads, and the derivatized beads were reacted with the oxidized form of horseradish peroxidase and 4-aminoantipyrine to give a surface-bound, red quinoneimine dye. The surface color was quantitatively determined by diffuse reflectance spectrophotometry. Hydrogen peroxide, which oxidizes the enzyme, was determined with a linear range of 10 to 200 μM and a standard deviation of ±10 μM. Samples containing glucose were treated with glucose oxidase to convert the glucose to hydrogen peroxide. Glucose standards showed a linear range of 50 to 200 mg/dL and a standard deviation of ±9 mg/dL. Results for control sera were in good agreement with values obtained by the usual aqueous method. After correcting for the blood hematocrit, glucose concentrations found for paired plasma and whole blo...

Homocapsaicin: it's NOT the 7-ene-9-methyl isomer.

http://dx.doi.org/10.1016/j.foodchem.2015.02.131 0308-8146/ 2015 Elsevier Ltd. All rights reserved. Dear Sir, Researchers analyzing foods containing capsaicinoids continue to perpetuate errors, most notably for the compound homocapsaicin, errors that I attempted to correct with my 2006 paper: ‘‘Homocapsaicin: nomenclature, indexing, and identification’’, Flavour and Fragrance Journal 2007, 22, 243–248. The three homocapsaicin isomers mentioned in the literature are listed in the table below. Research on the capsaicinoids continues to be very active: nearly 3600 citations that mentioned capsaicin or capsaicinoids were published between the years 2012 and 2014 (SciFinder search). Thirteen patents and papers in English mentioned the minor capsaicinoid homocapsaicin. Of the six references that described or depicted a structure for homocapsaicin, NONE gave the structure of one of the two known, naturally-occurring