Assay method

Several methods, physical and chemical, have been proposed for the estimation of the tocopherols. The proper estimation is usually preceded by separation in order to eliminate interfering substances or to separate the individual tocopherols. 

Tocopherols are converted by various oxidizing agents to the corresponding paraquinones (tocophcrylquinones) (III). 

The oxidizing agents used include gold chloride (potentiometric determination of the end point of the extremely slow oxidation) (Karrerefal, 1938) ceric sulfate (determination of the end point of the almost immediate reaction, with diphenylamine as indicator) (Kofler, 1941) lead tetraacetate (Kofler, 1941) but mostly ferric chloride in presence of a Fe -complex-forming compound, such as a,a -dipyridyl (Emmerie and Engel, 1938). The red color produced in this reaction is used for colorimetric determination. 

In the absence of acids the primary product of Emmerie and Engel s reaction with a-tocopherol is a compound called tocopheroxide (Harrison et al, 1956) which, according to Martins and Eilingsfeld (1957a,b) has the structure (IV) of a 6-quinone-9-acetal. 

On the addition of acids, this compound is transformed into tocopheryl-quinone. 

United State Pharmacopeia XX describes the following assay methods for acetylcholine as a reagent [11]  

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Assay Methods for Evaluating Progestin Agonists and Antagonists. 

Assay methods for j iZ-butyUithium essentiaUy are identical to those Hsted for //-butyUithium, except that the titrations are performed at lower temperatures because of the lower thermal stabUity of j iZ-butyUithium. 

A total material balance assay is a Fischer assay in which the retort gases are collected. A complete material balance closure and yields in excess of those expected from Fischer assay results are achieved. More complete descriptions of both the Fischer assay and the Tosco material balance assay methods have been reported (9). 

The hberated iodine, as the complex triiodide ion, may be titrated with standard thiosulfate solution. A general iodometric assay method for organic peroxides has been pubUshed (253). Some peroxyesters may be determined by ferric ion-catalyzed iodometric analysis or by cupric ion catalysis. The latter has become an ASTM Standard procedure (254). Other reducing agents are ferrous, titanous, chromous, staimous, and arsenite ions triphenylphosphine diphenyl sulfide and triphenjiarsine (255,256). 

Assay Methods. The primary assay for the streptovaricins is the microbiological assay using the agar diffusion method or a turbidimetric procedure (60). The streptovaricins can also be identified by paper (60,88) or thin-layer chromatography (3). 

In a fire-assay method used at the smelters, a weighed quantity of concentrate is mixed with sodium cyanide in a clay or porcelain cmcible and heated in a muffle furnace at red heat for 20—25 min. The tin oxide is reduced to metal, which is cleaned and weighed. Preliminary digestion of the concentrate with hydrochloric and nitric acids to remove impurities normally precedes the sodium cyanide fusion. 

An automated system for clinical analysis consists of the instmment (hardware), the reagents, and the experimental conditions (time, temperature, etc) required for each deterrnination. The reagents plus the experimental conditions are sometimes referred to as the chemistry of the system. The chemistry employed is generally similar to that used in manual assays because most automated assay methods have been adapted from the manual ones. However, automated analy2ers rarely afford the flexibiUty of experimental procedure that is possible in manual analysis. 

The assay methods Hsted in Table 1 for the various biochemical species can be classified according to reaction rate behavior, eg, end point vs kinetic methods, blanking schemes, or reaction principle and type of reagents employed. 

An assay method for benzaldehyde involves condensing benzaldehyde with hydroxyl amine hydrochloride to form an oxime. The released hydrochloric acid is then titrated. 

Benzaldehyde is sold as technical grade or as meeting the specifications of the NationalVormulary (NF) (7), the Vood Chemicals Codex (FCC) (8), or the British Pharmacopeia (BP) (9) (Tables 4 and 5). The test methods used for the analysis of benzaldehyde are standard methods, with the exception of the assay method. 

The assay method involves the reaction of benzaldehyde with hydroxylamine hydrochloride in an alcohoHc solution. Benzaldehyde oxime, water, and hydrochloric acid are the products of the reaction. The hydrochloric acid formed is then titrated with standard caustic solution to determine the benzaldehyde assay. 

In biological materials, various nonspecific precipitants have been used in the gravimetric deterrnination of choline, including potassium triiodide, platinum chloride, gold chloride, and phosphotungstic acid (28). Choline may also be determined spectrophotometricaHy and by microbiological, enzymatic, and physiological assay methods. 

Comparing Assays/Methods with a Range of Ligands... 

Table XII. Microbiological Assay Methods First Used to Determine Amino Acids...

Activation procedures (Shimomura, 1989). To activate trace amounts of panal, several granules of the crystals of ammonium sulfate are added to 5-50 il of a sample made in 30% methanol, making sure that (NH4)2S04 is in excess to saturate the solution. The mixture is left standing at room temperature for 1 h (85% activation) or one night (complete activation). After the activation, 1 jig of panal emits about 1.5 x 1010 photons using the assay method given below. 

Assay methods. Activity can be measured at pH 5.5 or at pH 8.0. With the same sample, the pH 5.5 method gives a much higher luminescence intensity than the pH 8.0 method (and, shorter reaction time), although the total amounts of light emitted by the two methods are practically equal (Fig. 9.4). The pH 5.5 method is susceptible to inhibition by various salts, whereas the pH 8.0 method is not affected. 

Activation of the M. citricolor luciferin precursor with decylamine. The following mixture was incubated overnight at room temperature (23 24°C) luciferin precursors before the separation of isomers by F1PLC (50 pi, activity 2 x 1012 photons by the assay method described above), 30 mM Bis-tris buffer (pH 6.4, 4.5 ml) acetonitrile... 

Aristostomias, 328-330, 338 Armillaria, 267, 271, 333 Arrow worm (Chaetognath), 161 Arthropoda, 1, 47 Ascomycetes, 266 Ascorbate, 187-191, 194-196,198 Aspisoma, 337 Assay methods... 

Various assay methods have been used to detect the presence of inhibitory substances. These include some of the classical tests used by investigators of growth-promoting substances—i.e., the various Avena coleoptile assays which utilize intact, decapitated, or isolated cylinders and the split pea stem test. Effects on seed germination and seedling shoot or root growth and development have also been measured in addition to other visible expressions of inhibition. Details of many of these tests have been compiled by Mitchell et al. (99). Tests have been carried out in Petri dishes, with various solution culture techniques, and by sand and soil culture. Effects so measured may or may not be similar to those obtained under field situations— i.e., the establishment of inhibition under controlled conditions pro-... 

Macarron R, Hertzberg RP (2002) Design and implementation of high throughput screening assays. Methods Mol Biol 190 1-29... 

Development of Assay Method for Ethylene-dinitramine , PATR 1200 (1942) 19) R. 

Assay Methods. A Na carbonate fusion prod is currently used for assay of mil grade AP (see below under Specifications), but recently attempts have been made to replace it with a method which is faster and simpler to carry out. Two procs have been proposed a) Titanous Chloride Titration. A measured excess of Ti trichloride is added to a known weight of AP which reacts according to the equation ... 

M. Roth, Evaluation of Assay Methods for Ammonium Perchlorate , ICRPG Round Robin No 28, PATR 3498 (1966) 16, AD 645935... 

Current analytical methods have difficulty detecting picogram levels of nucleic acids, particularly when high levels of other biopolymers (e.g., proteins) are present. The most widely used assay method employed by the pharmaceutical industry involves a nick translation DNA hybridization method (1). This assay offers high sensitivity and selectivity but has a number of drawbacks. 

Of the various chemical assays that have been developed for the saxitoxins (75,76), that described by Bates and Rapoport, based on the oxidation of saxitoxin to a fluorescent derivative, has proved to be the most useful. Other assay methods have been developed from it (77-79). The Bates and Rapoport method is virtually insensitive to the N-l-hydroxyl saxitoxins as originally described and so, like the presently available immunoassays, fails as a general assay for either concentration or toxicity. However, it is quite sensitive for those toxins it does detect and has been the basis for other useful methods. 

Urinary alkaline phosphatase activity. II. An analytical validation of the assay method. JAMA (1963), 185, 953-957. 

Table 15.3 lists the immimosera for which there is a need, or a potential need, today and indieates the reqnired potencies of these immimosera and the sahent features of the potency assay methods. 

Immunoglobulin Potency assay method Potency requirement... 

HPLC, high-petfomtance liquid chromatogtaphy ELISA, enzyme-linked immunosorbent assay method RIA, radioimmunoassay method. 

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