Assay for total citrate—
Standard preparations—
Dissolve a suitable quantity of citric acid, previously dried at 90
for 3 hours and accurately weighed, in water to obtain a stock solution having a known concentration of about 1.0 mg of anhydrous citric acid per mL. Pipet aliquots of 8, 9, 10, 11, and 12 mL, respectively, of the stock solution into separate 100-mL volumetric flasks, dilute with water to volume, and mix.
Assay preparation—
Pipet 10 mL of Solution into a 100-mL volumetric flask, dilute with water to volume, and mix. Pipet 5 mL of this solution into another 100-mL volumetric flask, dilute with water to volume, and mix.
Procedure—
Pipet 1 mL of the
Assay preparation, each
Standard preparation, and water into a suitable test tube. To each tube add 1.3 mL of pyridine, and mix by swirling. To one tube at a time add 5.7 mL of acetic anhydride, and mix, using a rotary vortex stirrer. Immediately place in a water bath maintained at 31 ± 1.0
, and allow the color to develop for 33 ± 1 minutes. Determine the absorbance against the reference blank in 2.5-cm cells at 425 nm, taking care to measure the absorbance of each solution at the same elapsed time from mixing. Plot the absorbances of the
Standard preparations versus the concentrations, and draw the straight line best fitting the plotted points. From the graph so obtained, calculate the total citrate content, in mg per mL, of the Anticoagulant Citrate Phosphate Dextrose Solution taken by the formula:
0.2C,
in which
C is the concentration, in µg per mL, of anhydrous citric acid read from the standard curve.
Assay for total phosphate [expressed as monobasic sodium phosphate, monohydrate (NaH2PO4·H2O)]—
1
,2,4-Aminonaphtholsulfonic acid solution—Dissolve 0.5 g of 1,2,4-aminonaphtholsulfonic acid in about 150 mL of sodium metasulfite solution (3 in 20) in a 200-mL volumetric flask, warming if necessary. Add 5 mL of sodium sulfite solution (1 in 5), mix, and dilute with sodium metasulfite solution (3 in 20) to volume.
Standard preparation—
Dissolve about 0.44 g of monobasic potassium phosphate, accurately weighed, in water to make 1000 mL, and mix. Pipet 25 mL of this solution into a 100-mL volumetric flask, and dilute with water to volume so as to obtain a solution having a known concentration of about 0.11 mg per mL of monobasic potassium phosphate.
Assay preparation—
Dilute 5.0 mL of Solution with water to 100 mL.
Procedure—
Pipet 5 mL of
Standard preparation, 5 mL of
Assay preparation, and 5 mL of water, to provide a control, into separate 25-mL volumetric flasks. Treat the contents of each flask as follows. Add 10.0 mL of 1 N sulfuric acid, and mix. Add 2.0 mL of ammonium molybdate solution (1 in 40), and mix. Add 1.0 mL of
1,
2,
4-Aminonaphtholsulfonic acid solution, dilute with water to volume, again mix, and allow to stand for 10 minutes at 20
to 25
. Determine the absorbances of the solutions from the
Standard preparation and the
Assay preparation against the reference solution in 1-cm cells at 660 nm, with a suitable spectrophotometer, taking care to measure the absorbance of each solution at the same elapsed time from mixing. Calculate the quantity, in mg, of monobasic sodium phosphate (NaH
2PO
4·H
2O) in each mL of the Solution taken by the formula:
20.28C(AU / AS),
in which
C is the concentration, in mg per mL, of KH
2PO
4 in the
Standard preparation; and
AU and
AS are the absorbances of the solutions from the
Assay preparation and the
Standard preparation, respectively.
Assay for dextrose—
Tare a clean, medium-porosity filtering crucible containing several carborundum boiling chips or glass beads. Pipet 50 mL of freshly mixed alkaline cupric tartrate TS into a 400-mL beaker. Add the boiling chips or glass beads from the tared crucible, 45 mL of water, and 5.0 mL of Solution to the beaker. Heat the beaker and contents over a burner that has been adjusted to cause boiling of the solution to start in 3.5 to 4 minutes. Boil the solution for 2 minutes, accurately timed, and filter immediately through the tared crucible, taking care to transfer all of the boiling chips or glass beads to the crucible. Wash the precipitate with hot water and 10 mL of alcohol. Dry the crucible and contents at 110
to constant weight. Perform a blank determination, and correct the weight of the precipitate from the sample for any precipitate obtained in the blank. Each mg of cuprous oxide precipitate obtained from the substance under assay is equivalent to 0.496 mg of C
6H
12O
6·H
2O.
161
).