Phosphate esters detection

Instead of immobilizing the antibody onto the transducer, it is possible to use a bare (amperometric or potentiometric) electrode for probing enzyme immunoassay reactions (42). In this case, the content of the immunoassay reaction vessel is injected to an appropriate flow system containing an electrochemical detector, or the electrode can be inserted into the reaction vessel. Remarkably low (femtomolar) detection limits have been reported in connection with the use of the alkaline phosphatase label (43,44). This enzyme catalyzes the hydrolysis of phosphate esters to liberate easily oxidizable phenolic products. 

A triaryl phosphate ester, tributoxyethyl phosphate, was detected at a mean concentration of 11.3 ng/g in 41 of 115 human adipose tissue samples taken from cadavers from Kingston and Ottawa, Canada (LeBel and Williams 1986). Because triaryl phosphate esters have been found in Canadian drinking water and... 

A component of Skydrol 500B and Skydrol LD (tributyl phosphate) was detected in the air at the CP air test facility at Vancouver International Airport at a concentration of 0.04-0.3 mg/m3 (Labour-Canada 1990), indicating that organophosphate ester hydraulic fluids maybe released during aircraft maintenance operations on equipment using organophosphate ester hydraulic fluids. 

Confirmation by GC/MS is recommended. Recovery is acceptable (82%). Tributyl phosphate and triphenyl phosphate contamination was reported in procedural blanks and may prevent determination of phosphate esters at levels near the detection limit (1 ng/g) (LeBel and Williams 1983 1986). 

Biological. Incubation of C-ring labeled endothall (10 mg/mL) by Arthwbactersp., which was isolated from pond water and a hydrosol, in aerobic sediment-water suspensions revealed that after 30 d, 40% evolved as CO2. Glutamic acid was the major transformation product. Minor products were alanine, citric, and aspartic acids and unidentified products, some of which were tentativeiy identified as phosphate esters (Sikka and Saxena, 1973). In pond water treated with endothall (2 and 4 ppm), detectable levels were found after 7 d (Sikka and Rice, 1973). Biodegradation was rapid in an Ontario soil sample. After 1 wk, 70% of endothall added was converted to carbon dioxide (Simsiman et al, 1976). 

Thiamine shows a pH-dependent UV absorbance range of 230-270 nm. However, its UV absorbance is prone to interference by other endogenous UV absorbers in foods, such as nucleic acids (67,68). In a recent interlaboratory comparison of thiamine methods (42), the results obtained from an HPLC method using UV absorbance detection were rejected due to the presence of peaks that interfered with thiamine. In the interests of increased sensitivity and selectivity, the thiamine vitamers are generally converted to their thiochrome derivatives by alkaline oxidation and determined fluorimetrically (42,70). The thiochrome derivatives of thiamine and its phosphate esters all fluoresce at nearly identical excitation (365-375 nm) and emission (425-435 nm) maxima at pH over 8. The thiochrome derivatives are all relatively stable in alkaline solution at pH greater than 9 and room temperature. 

Donner, R. and Lohs, K., Cobalt chloride in the detection of organic phosphate ester by paper and especially thin-layer chromatography, J. Chromatogr., 17, 349, 1965 Chem. Abs., 62, 13842d, 1965. 

The problems associated with the separation of phosphate esters are the tendency of the spots to diffuse difficulty of reproducing RF values variability of RF values with complexity of the mixture being resolved and distance of solvent travel proper purification and equilibration of the filter paper effect of inorganic ions in natural mixtures and the choice of suitable solvent mixtures for desired separations. The problem of detecting the separated spots is somewhat simpler. In addition to methods applicable to the detection of reducing sugars (e.g., use of aniline salts fructose esters are detected by naphthoresorcinol-acid), methods depending on phosphomolybdate formation are most commonly used. Hanes and Isher-... 

The Lobry de Bruyn reaction can be carried out on the paper, followed by chromatography of the products.167 168 The procedure of Bayly and Bourne22 for forming benzylamine derivatives of oligosaccharides on filter paper has been mentioned previously. Enzymic hydrolysis of phosphate esters, in conjunction with paper chromatography, aids detection of the spots.126... 

When lecithin is hydrolyzed in acidic medium, both the fatty acid ester bonds and the phosphate ester bonds are broken and free fatty acids and inorganic phosphate are released. Using a molybdate test, we can detect the presence of phosphate in the... 

Houten KAV, Heath DC, Pilato RS. Rapid Luminescent detection of phosphate esters in solution and the gas phase using (dppc)Pt S2C2(2-pyridyl)(CH2CH2OH). J Am Chem Soc 1998 120 12359-60. 

When immobilized in a polymer-plasticizer matrix, (dppe)Pt S2C2(2-py) (CH2CH2OH), can be used for the rapid selective detection of volatile fluoro-, chloro-, and cyanophosphates (Table III, Fig. 10). As with many film immobilized lumiphores, the reactivity is controlled by the immobilizing matrix (39-44, 46, 50-52, 57, 58, 63, 75-77) Such an effect is seen as the sensitivity to phosphate esters increases as plasticizer (triethyl citrate, TEC) concentration increases in the cellulose acetate CA/TEC film (Table IV). 

The urine of dystrophic patients was reported in 1949 by Minot et al. (Mil) to contain quantities of organic ribose phosphate esters, which could be detected for diagnostic purposes by a modified qualitative Benedict s test (03). Walton and Latner (W5), however, found this proposed test to have no diagnostic value. Determined and thorough biochemical investigations in large numbers of patients with all types of muscular dystrophy (Pll, RIO) have since shown that any difference... 

Proof that a lysine residue has been modified can be readily obtained because pyridoxyl derivatives of lysine possess characteristic white-blue fluorescence (Ronchi et al. 1969). In addition, they have a distinctive absorption maximum at 325 nm with of 9710 cm (Fisher et al. 1963). Finally, a radiochemical label can be introduced by reducing the pyridoxal-5-phosphate protein complex with tritium-labelled sodium borohydride. The peptide containing the derivatized lysine can therefore be detected either by fluorimetry, spectrophotometry or radiochemical techniques following routine procedures of proteolytic digestion and fractionation. Acid hydrolysis in 6 N HCl for 24 hr of peptides containing pyridoxal-5-phosphate lysine yields pyridoxyl-lysine since phosphate esters are readily hydrolyzed under these conditions. Pyridoxyl-lysine is eluted between lysine and histidine from a 55 cm column of Beckman 50 resin with 0.15 M citrate buffer pH 5.28. 

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