5. AOACSPDSMethods-2018AwardsV3

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separate centrifuge tubes. The samples should be prepared in duplicate. These types of samples typically are of very high molecular weight and require longer dissolution times (Table 2018.14C ). G. System Suitability ( a ) Prepare a 1 mg/mL solution in water of gum acacia to be used as the negativecontrol. ( b ) The gum acacia must show no response. H. Determination ( a ) Colorimetric reaction.–To each calibration, system suitability and sample solutionadd 2.0 mL of alkaline hydroxylamine hydrochloride. Vortex for 5 s and allow the solutions to react at room temperature for 5 min. Add the amount of hydrochloric acid as determined in the optimization of pH and vortex for 5 s. Add 1.0 mL of ferric chloride and vortex for 5 s. At this point, the solution will turn a reddish- orange color. ( b ) Small gas bubbles will form which can be removed by sonication for a few seconds. If the solutions sit around for more than 10 min, sonication may have to be repeated to remove residual air bubbles. ( c ) Measurement.–After the color has developed for 5 min, but no longer than60 min, zero the spectrophotometer at 540 nm with the blank standard. Measure the absorbance of the standards and samples. Report the mean of the duplicate measurements. I. Calculations ( a ) Stock standard solution.-Calculate the concentration of the acetylcholine chloride in the stock standard solution as follows: Stock standard solution, mg/mL = M x P / 50 ( b ) Calibration standard solutions.–Using the concentration of the acetylcholine chloride stock solution, calculate the actual mg of each standard solution asfollows: Calibration standard solution, mg = S x V where S is the concentration of the stock solution (mg) calculated from (a) and V is the volume of stock solution pipetted (0.2. 0.4, 0.6, 0.8, 1.0 mL) ( c ) Calibration curve. – Plot absorbance at 540 nm vs mg of calibration standards. Using linear regression calculate the slope, y-intercept and r 2 of the curve. ( d ) System suitability. – The r 2 value of the calibration curve must be≥0.98. ( e ) Aloe vera polysaccharide content. – Calculate the aloe vera polysaccharide content in % w/w as follows: where M = mass of acetylcholine chloride (mg), P = purity of acetylcholine chloride

Total Aloe vera polysaccharides (w/w %) = [(A-b)/m] x [V/W] x 100