Assay preparation

Blinks, J. R., et al. (1978). Practical aspects of the use of aequorin as a calcium indicator Assay, preparation, microinjection, and interpretation of signals. Method. Enzymol. 57 292-328. 

Assay preparation Transfer an accurately measured volume of injection, equivalent to about 50 mg of miconazole, to a 100 mL volumetric flask, dilute with Mobile phase to volume, and mix. Transfer 10 mL of this solution to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix. 

Procedure (NOTE - Allow the chromatograph to run for at least 16-18 min between injections to allow for elution of all components associated with the injection vehicle.) Separately inject equal volumes (about 20 pL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of Ci8H14CL4N20 in each milliliter of the injection given by the formula ... 

Assay preparation Transfer an accurately weighed portion of topical powder, equivalent to about 20 mg of miconazole nitrate, to a stoppered 50-mL centrifuge tube. Add 25 mL of methanol, and shake by mechanical means for 30 min to dissolve the miconazole nitrate. Centrifuge to obtain a clear supernatant liquid. Transfer 5 mL of this solution to a test tube, add 2 mL of Internal standard solution and evaporate at a temperature not higher than 40 °C with the aid of a current of nitrogen to dryness. Dissolve the residue in 2 mL of a mixture of chloroform and methanol (1 1). 

The retention time ratio of the valproic acid peak to the internal standard peak is obtained from the standard preparation and the assay preparations. 

Assay preparation. Transfer not less than 20 Capsules to a blender jar or other container, and add about 150 mL of methylene chloride, and cool in a solid carbon dioxide acetone mixture until the contents have solidified. If necessary, transfer the mixture of capsules and methylene chloride to a blender jar, and blend with high-speed blender until all the solids are reduced to fine particles. Transfer the mixture to a 500-mL volumetric flask, add n-heptane to volume, mix, and allow solids to settle. Transfer an accurately measured volume of this solution, equivalent to 250 mg of valproic acid, to a 100 mL volumetric flask, dilute with w-heptane to volume, and mix. Transfer 5.0 mL to a container equipped with a closure. Add 2.0 mL of the internal standard solution, close the container, and mix. 

Assay preparation. Transfer an accurately measured volume of syrup, equivalent to about 250 mg of valproic acid, to a separator. Add 40 mL of water and 2.0 mL of hydrochloric acid not less than 20 capsules to a blender jar or other container, and add about 150 mL of methylene chloride, and cool in a solid... 

For the tumor spheroid-based migration assay, prepare the EC... 

Assay preparation Transfer approximately 0.24 g of sorbitol to a 50 mL volumetric flask, dissolve in a 10 mL of water, dilute with water to volume and mix. 

To set up the inhibition assay, prepare a table similar to Table E5. 1. Inhibitor should appear in the list of reagents before tyrosinase. Use the same level of tyrosinase and the same dopa stereoisomer as in part C. Vary the amount of dopa as in part C. A constant amount of inhibitor (cinnamic acid or thiourea) should be added to each cuvette. You will have to determine this level of inhibitor by trial and error. The desired inhibition rate with saturating substrate is about 50% of the uninhibited rate. Add all reagents except tyrosinase, mix well, and determine the blank rate, if any. Add tyrosinase, mix, and immediately record AA75 for 2 minutes. From recorder traces or graphs of A475 vs. time, calculate AA/min for each assay. 

Assay preparation Transfer about 100 mg of Omeprazole, accurately weighed, to a 50-ml volumetric flask, dissolve in and dilute with Diluent to volume, and mix. Transfer 5 ml of this solution to a 50-ml volumetric flask, dilute with Diluent to volume, and mix. 

Identification The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay. 

Procedure After 2 h, remove each sample from the basket, and quantitatively transfer into separate volumetric flasks to obtain a solution having a final concentration of about 0.2 mg/ml. Proceed as directed for the Assay preparation in the Assay, starting with "Add about 50 ml of Diluent. Calculate the quantity, in mg, of omeprazole (C17H19N3O3S) dissolved in the Medium by the formula ... 

Assay preparation Weigh and mix the contents of not fewer than 20 Capsules. Transfer an accurately weighed portion of the mixture, equivalent to about 20 mg of omeprazole, to a 100-ml volumetric flask, add about 50 ml of Diluent, and sonicate for 15 min. Cool, dilute with Diluent to volume, mix, and pass through a membrane filter having 0.45 /im or finer porosity. [Note Bubbles may form just before bringing the solution to volume. Add a few drops of dehydrated alcohol to dissipate the bubbles if they persist for more than a few minutes]. 

High-scoring compounds are then prepared and screened in a biochemical assay. Preparing requires either synthesizing or purchasing a sample of the compound. Sample purchase is normally much faster than synthesis. For this reason, compounds in a virtual screen are often limited to structures that can be purchased from any number of commercial suppliers. Databases of available molecules have been prepared for the sole purpose of assisting virtual screening. 

Where Au = area under the peak relating to the Assay Preparation... 

Assay Preparation - Transfer about 30 mg of Cholecal-ciferol, accurately weighed, to a low-actinic, 25-ml volumetric flask, and proceed as directed for Standard Preparation, beginning with "add isooctane to volume," to obtain a solution having a concentration of about 120 pg per ml. 

Procedure - Equilibrate the Standard Preparation and the Assay Preparation in the dark at 80° for 2.5 hours, accurately timed. Cool, and introduce equal volumes (5 to 10 1) of the heat-equilibrated Standard Preparation and Assay Preparation into the high-pressure liquid chromatograph (See chromatography <621>) by means of a suitable sampling valve. Measure the peak responses obtained for the Assay Preparation and the Standard Preparation, and calculate the quantity, in mg, of C27H44O in the portion of Cholecalciferol taken by the formula 0.25C(Ay/Ae)> in which C is the concentration, in g per ml, of USP Cholecalciferol RS in the Standard Preparation, and Ay and As are the peak responses for cholecalciferol obtained for the Assay Preparation and the Standard Preparation, respectively. 

Procedure Separately inject equal volumes (about 20 pL) of the Standard Preparation and the Assay Preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. 

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