Separation and Assay Methods

Physical Methods and Physical Chemistry.—Separation and Assay Methods. A procedure for h.p.l.c. of plant pigments has been used to separate the carotenoids of spinach and of a diatom. H.p.l.c. separations of citrus carotenoids, and of retinal (67) isomers have also been reported. Carotenoid mixtures have also been separated efficiently and rapidly by centrifugal chromatography. ... 

Separation and Assay Methods. The g.l.c. behaviour of the hydrogenation products of about sixty carotenoids, carotenoid acetates and trimethylsilyl ethers, and triterpenoid carotenoids has been studied extensively. The relative retention times of these compounds in three systems at different temperatures and with temperature... 

Separation and Assay. Methods have been described for the h.p.l.c. and m.s. determination of ubiquinone homologues in animal tissues,h.p.l.c. analysis of ubiquinone and phylloquinone in blood,of menaquinone-4 (230), 2,3-epoxymenaquinone-4 (231), and 4-demethylmenaquinone-4 (232) and of phylloquinone and menaquinone-4. The polarographic determination of phylloquinone and menaquinone-4 has been reported. 

In the late 1950s and early 1960s several papers appeared which documented the decrease of alkaloid content during plant development. Sometimes periodic variations (increases or decreases) were also observed. Modem separation and assay methods made these experiments possible. 

Armstrong WG (1968) A method for the simultaneous separation and assays of peptides and attached carbohydrate and fluorescent components. Automat Anal Chem (Technicon Symp 3rd, 1967) 1, 295-299. 

Many methods have been developed for the quantitative determination of each class of surfactants. The analysis of commercial surfactants is much more complicated since they may be comprised of a range of compounds within a given structural class, may contain surface-active impurities, may be formulated to contain several different surfactant classes, and may be dissolved in mixed organic solvents or complex aqueous salt solutions. Each of these components has the potential to interfere with a given analytical method so surfactant assays are sometimes preceded by surfactant separation techniques. Both the separation and assay techniques can be highly specific to a given surfactant/solution system. Table 3.4 shows some typical kinds of analysis methods that are applied to the different surfactant classes. 

Separation and Assay. H.p.l.c. methods have been described for the determination of chloroplast pigments,phytoplankton pigments,and provitamin A carotenoids in tomatoes.Other chromatographic procedures have been devised for the separation of Capsicum carotenoids and chloroplast pigments from tobacco mutants, " and methods for the high-speed video-densitometric determination of carotenoids separated by t.l.c., and for the dual assay of carotenoids and vitamin A in human liver have been reported. H.p.l.c. has been used for the separation of cis-trans-isomers of retinal, retinol and retinyl... 

Degraded Carotenoids Physical Methods Separation and Assay N.M.R. Spectroscopy Mass Spectrometry Chiroptical Methods Electronic Absorption Spectroscopy Infrared and Resonance Raman Spectroscopy Other Spectroscopic Techniques Miscellaneous Physical Chemistry Photoreceptor Pigments Biosynthesis and Metabolism Stereochemistry Enzyme Systems Inhibition and Regulation... 

THE FACTORS AND THEIR LEVELS. EXAMINED IN THE ROBUSTNESS TEST OF (82) ON A CHROMATOGRAPHIC METHOD FOR THE SEPARATION AND ASSAY OF A DRUG SUBSTANCE AND TWO RELATED COMPOUNDS IN TABLETS... 

The second phase is the determination of the number or percentage of true positive results achieved with the test in a population of animals that have been dosed with the compound of interest. This is an essential phase in the development of residue methods and the rigorous assessment of the true positive rate requires confirmation by a separate accepted assay method(s). In addition, part of this study may need to be performed under field conditions, particularly if the test is intended to be used in a non-laboratory environment. 

One of the more impressive advantages of LC/MS is that less resolution and selectivity are required from the LC separation. For assay methods involving complex matrices such as plasma, the separation of analyte from matrix components is not as critical, so the separation can be much shorter. Assay methods requiring 15 to 20 min with LC/UV or fluorescence can be shortened to only a few minutes. Methods that previously required large numbers of theoretical plates and long (25-cm) analytical columns can now be implemented with less resolution and shorter (1- to 15-cm) columns. 

Physical Methods.—Separation and Assay. A range of isomers of astaxanthin (8) diacetate (9-cis, 13-cis, 15-cis, 9,9 -di-cis, 9,13-di-cw, 9,13 -di-cw, 13,13 -di-cw, 13,15-di-cw), prepared by thermal and iodine-catalysed isomerization of irans-(S) have been separated by h.p.l.c.126 A procedure has been developed for separation of bean leaf etioplast pigments, including carotenoids,127 by h.p.l.c. H.p.l.c. separations of esters of all-trans-, 9-cis-, 11-cw-, and 13-cw-retinol,128-130 and determinations of retinol in serum,131 retinol and 13-cw-retinoic acid,132 and the aromatic retinoid (195)133 in plasma have been described. A reversed-phase ion-pair... 

Physical Methods. H.p.l.c. procedures for the separation and assay of ubiquinone and homologues278-280 and of menaquinone cis- and fra/ts-isomers, 2,3-epoxides, and chain-length homologues281 282 have been described. A XH n.m.r. study has been reported283 of the location and motion of ubiquinones in perdeuteriated phosphatidylcholine bilayers. Other aspects of the interaction of ubiquinone with phospholipid monolayers have been studied.284... 

One of the problems of barbiturate analysis is the isolation of the compound from biological material.427,428,437,449,513 Extraction is the most accepted method, but some assays take advantage of omitting the isolation and purification step. Special attention was devoted to separation and assay of particular barbiturate drugs in combination with other compounds. For example, many methods were devised for the determination of phenobarbital and hydantoin used together in anticonvulsive therapy.514 Several procedures were proposed for the determination of barbiturates along with their metabolites in body fluids.437... 

Physical Methods.—Separation and Assay. Increasing use is being made of h.p.l.c. in the carotenoid field. This technique will clearly become the method of choice for carotenoid separation, purification, and analysis. The first papers on carotenoid h.p.l.c. have recently been published. These include a systematic study of the chromatography of model mixtures of carotenes, carotenediols, cis trans isomers, and diastereoisomers. ° Many papers report the use of h.p.l.c. 

Separation and Assay. Procedures for the separation, purification, and assay of carotenoids and retinoids by h.p.l.c., g.c., and g.c.-m.s. are given in an extensive article." Another, general, review includes information on the h.p.l.c. separation of retinoids.A particularly useful method has been developed for resolution and analysis of some carotenoid optical isomers.For example, (3R,3 R)-, (3S,3 S)-, and (3/ ,3 5)-astaxanthin were converted into the diastereomeric (-)-camphanic acid diesters, which were separated by h.p.l.c. This procedure has been used to analyse the isomeric composition of a natural astaxanthin sample. An h.p.l.c. procedure for separation of a-, P-, and y-carotenes (173)—(175) and lycopene (176) has been described." Several papers describe methods for the h.p.l.c. separation and purification of various retinal and retinol isomers and derivatives.A procedure for the preparative t.l.c. of oxidation products of retinyl acetate has been described,and a competitive protein-binding radioassay for retinoic has been reported. 

The average composition of copolymers can be determined most readily for the case where the monomeric units can be isolated and identified by suitable scission or degradation reactions. This is the usual method for the elucidation of protein structure. The proteins are hydrolyzed by acids and / or bases in an automated apparatus. The resulting amino acids are chromato-graphically separated and assayed quantitatively via the color reaction with ninhydrine in so-called amino acid analyzers. 

The fire-assay method described above has some advantages as the separation and preconcentration method of precious metals. It is useful where large samples are required owing to their extremely low... 

HIBA/sodium octanesuIfonate-a-HIBA systems were employed for separation and assay of lanthanide fission products. In one of the studies, the HPLC method was used to separate and estimate a lanthanide fission product, e.g., lanthanum and a heavy element, e.g., uranium. The bum-up obtained was found to be in good agreement with the conventional mass spectrometric method. In another study. 

Whitmore, D. A., K. P. Wheeler, Separation and assay of nonionic detergents, Biochem. Bio-phys. Methods, 1980,2,133-138. 

Analytical Techniques. Sorbic acid and potassium sorbate are assayed titrimetricaHy (51). The quantitative analysis of sorbic acid in food or beverages, which may require solvent extraction or steam distillation (52,53), employs various techniques. The two classical methods are both spectrophotometric (54—56). In the ultraviolet method, the prepared sample is acidified and the sorbic acid is measured at 250 260 nm. In the colorimetric method, the sorbic acid in the prepared sample is oxidized and then reacts with thiobarbituric acid the complex is measured at - 530 nm. Chromatographic techniques are also used for the analysis of sorbic acid. High pressure Hquid chromatography with ultraviolet detection is used to separate and quantify sorbic acid from other ultraviolet-absorbing species (57—59). Sorbic acid in food extracts is deterrnined by gas chromatography with flame ionization detection (60—62). 

Estimation of Other Alkaloids in Opium. Of the other alkaloids the most important is codeine, and processes for its estimation in opium have been described by Cespari, Andrews,and Annett and co-workers methods for its assay in admixture with other drugs in tablets and other products are also available. The estimation of papaverine has been described by Issekutz,i and of narcotine by Snesarov. As to methods for the separation and estimation of two or more of these subsidiary alkaloids, codeine and narcotine have been dealt with by van der Widen,narcotine and papaverine by Annett and Bose, ( and the bromination of codeine and narceine has been studied by Vaisberg et al. with a view to estimation by this means. 

Agarwal et al. 1978), the quantification of these specific enzymes may indicate that exposure to endosulfan has occurred. Blood tests, such as decay curves for aminopyrine in plasma, which are semiquantitative indices of liver enzyme induction, have been used successfully in the past to demonstrate enzyme induction in pesticide-exposed workers. Because numerous chemicals found at hazardous waste sites also induce these hepatic enzymes, these measurements are not specific for endosulfan exposure. However, measurements of enzyme activity, together with the detection of the parent compound or its metabolites in tissue or excreta, can be useful indicators of exposure. All of these potential biomarkers require further verification in epidemiological studies. Further studies with focus on the development of methods to separate and measure the estrogenicity of endosulfan in in vitro assays would be valuable since these assays are more sensitive and discriminative than other conventional biomarkers. Preliminary results have been presented by Sonnenschein et al. (1995). 

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