Internal standards, spectrophotometric assays

One milligram of microsomal protein is added to 0.1 M potassium phosphate buffer (pH 7.4) containing 50 mM NaF, 10 mM dithiothreitol, 1 mM EDTA, 20% glycerol (v/v), 150 iM 5-cholestene-3/3, 7a-diol, and 0.915% CHAPS. The reaction is initiated by 1 mM NAD+ to give a final reaction volume of 1.0 mL. After incubation at 37°C for 5 minutes, the reaction is terminated by adding 2 mL of 95% ethanol. An internal recovery standard, 4-cholesten-3-one (3 fig in methanol) is also added. The steroid products are extracted into 5 mL of petroleum ether (repeated twice). After the ether has been removed at 40°C under a stream of nitrogen, the products are dissolved in 100 fxL of mobile phase and 20 ju.L is injected into the column. The amount of product formed is linear with protein (to 1.5 mg) and with time (up to 10 min, 1 mg protein). The assay is much more sensitive than the direct spectrophotometric assay, and it avoids the use of thin-layer chromatography and radioisotopes described in other methods. 

The quantum yield of fluorescence from p-hydroxybenzoic acid is low (10). It was found that this fluorescence was subject to strong quenching in irradiated solutions. In the fluorescence assay, therefore, p-hydroxybenzoic acid was added as an internal standard. The G-values so obtained were in agreement with those obtained by spectrophotometric assay at an isobestic point. 

Since, In practice, the efficiency frequently varies, it is necesseuy to determine the counting efficiency before one can compare different samples. A radioactive standard of acciirately known activity is essential for the determination of the efficiency counting. The use of radioactive standards is in principle similar to that of standards in colorimetric or spectrophotometric assays. There are two types of standards (i) internal standard (this is usually a p-emitter of accurately known activity which, when dissolved in unquenched scintillation mixture, provides a reference standard), and (ii) external standard (ay-emitter incorporated in most instruments). 

The expert Committee on Biol( cal Standardization of the World Health Organization is proceeding with the establishment of an international standard for vitamin Bu. Until this standard is available Crystalline B 2 Merck is recommended as a standard. The U. S. P. identification and spectrophotometric assay for crystalline B12 are as follows (48) ... 

Prieto et al. (1999) reported another spectrophotometric method for the quantitative determination of antioxidant capacity based on the reduction of Mo(VI) to Mo(V) by vitamin E in acidic conditions with incubation at 95°C for 90 min. The subsequent green phosphate/Mo(V) complex, after cooling at room temperature was monitored at 695 nm with a calibration range of 0.2-2 x 10 M (r = 0.997) and a detection limit of 0.135 pmol vitamin E. The method was applied for measurement of total antioxidant capacity of plant extracts and to determine vitamin E in a variety of grains and seeds, including corn and soybean. The recovery of vitamin E from seeds was determined by supplementing the samples with the different vitamin E isomers or a-tocopherol acetate as internal standard and applying both the proposed method and a standard HPEC assay (Huo et al., 1996) and yielded a recovery of 93%-97% tocopherols. 

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