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ANTICOAGULANT AND PRESERVATIVE SOLUTIONS FOR HUMAN BLOOD

Category Anticoagulant for storage of whole blood.

 Anticoagulant and Preservative Solutions for Human Blood (Anticoagulant Citrate Dextrose Solution (ACD) and Anticoagulant Citrate Phosphate Dextrose Solution (CPD) are sterile and pyrogen-free solutions prepared with Water for Injection, filtered, distributed in the final containers and sterilized. The contents of sodium citrate dihydrate (C6H5Na3O7.2H2O), dextrose monohydrate (C6H12O6.H2O) or anhydrous dextrose (C6H12O6) and sodium dihydrogenphosphate dihydrate (NaH2PO4.2H2O) are not less than 95.0 per cent and not more than 105.0 per cent of that stated in the formulae below. The contents of citric acid monohydrate (C6H8O7.H2O) or anhydrous citric acid (C6H8O7) is not less than 90.0 per cent and not more than 110.0 per cent of that stated in the formulae below. Other substances, such as red-cell preservatives, may be included in the formula provided that their names and concentrations are stated on the label.

Requirements and advice concerning the containers are given in Appendix 11.3.

ANTICOAGULANT CITRATE DEXTROSE SOLUTION

Acid Citrate Dextrose Solution (ACD)

Description Colourless or faintly yellow, clear liquid, free from particles.

Packaging and storage Anticoagulant Citrate Dextrose Solution shall be preserved in tightly closed, tamperevident containers of plastic or other suitable materials.

Labelling Comply with the “General Information for Biological Products”, p. 177. In addition the label on the container states (1) the composition and volume; (2) where applicable, the maximum amount of blood to be collected in the container.

Identification

          A. Carry out the test as described in the “Thin-layer chromatography” (Appendix 3.1), using silica gel G as the coating substance and a mixture of 2 volumes of water, 3 volumes of methanol, 5 volumes of anhydrous glacial acetic acid and 10 volumes of 1,2-dichloroethane as the mobile phase. The volumes of solvents have to be measured accurately since a slight excess of water produces cloudiness. Apply separately to the plate 2 μl of each of the following solutions. For solution (A) dilute 2 ml of the solution being examined (for formula A) or 3 ml (for formula B) to 100 ml with a mixture of 2 volumes of water and 3 volumes of methanol. For solution (B1) dissolve 10 mg of Dextrose RS in a mixture of 2 volumes of water and 3 volumes of methanol and dilute to 20 ml with the same mixture of solvent. For solution (B2) dissolve 10 mg each of Dextrose RS, Lactose RS, Fructose RS, and Sucrose RS in a mixture of 2 volumes of water and 3 volumes of methanol and dilute to 20 ml with the same mixture of solvents. After removal of the plate, allow it to dry in warm air, repeat the development immediately, after renewing the mobile phase. Dry the plate in a current of warm air and spray evenly with a solution of 0.5 g of thymol in a mixture of 5 ml of sulfuric acid and 95 ml of ethanol, heat at 130º for 10 minutes and allow it to cool. The principal spot in the chromatogram obtained from solution (A) is similar in position, colour and size to the principal spot in the chromatogram obtained from solution (B1). The test is not valid unless the chromatogram obtained from solution (B2) shows four clearly separated spots.

B. To 2 ml add 5 ml of copper-citric TS and heat to boiling: an orange precipitate is produced and the solution becomes yellow.

C. To 2 ml (for formula A) add 3 ml of water or to 4 ml (for formula B) add 1 ml of water. It yields the reactions characteristic of citrates (Appendix 5.1).

D. It yields reaction B characteristic of sodium salts (Appendix 5.1).

pH 4.7 to 5.3 (Appendix 4.11).

Hydroxymethylfurfural To 2.0 ml add 5.0 ml of a 10 per cent w/v solution of p-toluidine in 2-propanol containing 10 per cent v/v of glacial acetic acid and 1.0 ml of a 0.5 per cent w/v solution of barbituric acid. The absorbance, determined at 550 nm after allowing the mixture to stand for 2 to 3 minutes, is not more than that of a standard prepared at the same time in the same manner using 2.0 ml of a solution containing 5 ppm of hydroxymethylfurfural for formula A or 3 ppm of hydroxymethylfurfural for formula B.

Bacterial endotoxins and Pyrogens Perform one of the following tests.

Bacterial endotoxins When tested as described in the “Test for Bacterial Endotoxins” (Appendix 8.5), it contains not more than 5.56 Endotoxin Units per ml.

          Pyrogens Complies with the “Pyrogen Test” (Appendix 8.2), using per kg of the rabbit’s weight 10 ml of a dilution in pyrogen-free saline TS containing 0.5 per cent w/v of sodium citrate dihydrate.

Sterility Comply with the “Sterility Test” (Appendix 10.1).

Assay

          FOR CITRIC ACID To 10.0 ml of Anticoagulant Citrate Dextrose Solution (for formula A) or to 20.0 ml (for formula B) add 0.1 ml of phenolphthalein TS. Titrate with 0.2 M sodium hydroxide VS until a pink colour is obtained. Perform a blank determination, and make any necessary correction. Each ml of 0.2 M sodium hydroxide is equivalent to 14.01 mg of C6H8O7.H2O or to 12.81 mg of C6H8O7.

          FOR SODIUM CITRATE Prepare a chromatographic column (10 cm × 10 mm) and filled with strongly acidic ion-exchange resin (300 to 840 μm). Maintain a 1-cm layer of liquid above the resin at all times. Wash the column with 50 ml of de-ionized water at a flow rate of 12 to 14 ml per minute.

          Dilute 10.0 ml of the solution being examined (for formula A) or 15.0 ml (for formula B) to about 40 ml with de-ionized water in a beaker and transfer to the column reservoir, washing the beaker three times with a few ml of de-ionized water. Allow the solution to run through the column at a flow rate of 12 to 14 ml per minute and collect the eluate. Wash the column with two 30-ml portions and with one 50-ml portion of de-ionized water. The column can be used for three successive determinations before regeneration with three times its volume of dilute hydrochloric acid. Titrate the combined eluate and washings (about 150 ml) with 0.20 M sodium hydroxide, using 0.1 ml of phenolphthalein TS as indicator.

          Calculate the content of sodium citrate in g per litre from the following expressions:

For formula A: 1.961n – 1.40C

or 1.961n – 1.53C’

For formula B: 1.307n – 1.40C

or 1.307n – 1.53C’

where n = number of ml of 0.20 M sodium hydroxide used in the titration,

C = content of citric acid monohydrate in g per litre determined as prescribed above,

C’ = content of anhydrous citric acid in g per litre determined as prescribed above.

          FOR REDUCING SUGARS Dilute 5.0 ml (for formula A) or 10.0 ml (for formula B) to 100.0 ml with water. Introduce 25.0 ml of the solution into a 250-ml conical flask with ground-glass neck and add 25.0 ml of copper-citric TS. Add a few pieces of porous material, attach a reflux condenser, heat so that boiling begins within 2 minutes, and boil for exactly 10 minutes. Cool and add 3 g of potassium iodide dissolved in 3 ml of water. Add 25 ml of a 25 per cent w/w solution of sulfuric acid with caution and in small quantities. Titrate with 0.10 M sodium thiosulfate using 0.5 ml of starch TS, added towards the end of the titration, as indicator (n1 ml). Carry out a blank titration using 25.0 ml of water (n2 ml).

          Calculate the content of reducing sugars as anhydrous dextrose or as dextrose monohydrate, as appropriate, from Table 1.

Table 1

ANTICOAGULANT CITRATE PHOSPHATE DEXTROSE SOLUTION

Citrate Phosphate Dextrose Solution (CPD)

Description; Packaging and storage; Labelling See under Anticoagulant Citrate Dextrose Solution, p. 183.

Identification

A. Complies with the tests for Identification described under Anticoagulant Citrate Dextrose Solution, p. 183.

B. It yields reaction B characteristic of phosphates (Appendix 5.1).

pH 5.3 to 5.9 (Appendix 4.11).

Hydroxymethylfurfural Complies with the tests for formula A described under Anticoagulant Citrate Dextrose Solution, p. 183.

Bacterial endotoxins and Pyrogens; Sterility Complies with the tests described under Anticoagulant Citrate Dextrose Solution, p. 183.

Assay

          FOR SODIUM DIHYDROGENPHOSPHATE DIHYDRATE Dilute 10.0 ml of Anticoagulant Citrate Phosphate Dextrose Solution to 100.0 ml with water. To 10.0 ml of this solution add 10.0 ml of nitro-vanado-molybdic TS. Mix and allow to stand at 20º to 25º for 30 minutes. At the same time and in the same manner, prepare a reference solution using 10.0 ml of a standard solution containing 0.219 g of potassium dihydrogenphosphate per litre. Measure the absorbance of each of the two solutions at 450 nm (Appendix 2.2), using as the blank a solution prepared in the same manner using 10 ml of water. Calculate the content of NaH2PO4.2H2O (P) in g per litre from the expression:

where C = concentration of potassium dihydrogenphosphate in the standard solution in g per litre,

A1 = absorbance of the test solution, and

A2 = absorbance of the reference solution.

FOR CITRIC ACID To 20.0 ml add 0.1 ml of phenolphthalein TS and titrate with 0.20 M sodium hydroxide. Calculate the content of citric acid monohydrate (C), or anhydrous citric acid (C’), in g per litre from the equations:

C = 0.7005n – 0.4490P

C’ = 0.6404n – 0.4105P

where n = number of ml of 0.20 M sodium hydroxide used in the titration, and

P = content of sodium dihydrogenphosphate dihydrate in g per litre determined as prescribed above.

          FOR SODIUM CITRATE Prepare a chromatographic column 0.10 m long and 10 mm in internal diameter and filled with strongly acidic ion-exchange resin (300 μm to 840 μm). Maintain a 1-cm layer of liquid above the resin at all times. Wash the column with 50 ml of de-ionized water at a flow rate of 12 to 14 ml per minute.

          Dilute 10.0 ml of the solution being examined to about 40 ml with de-ionized water in a beaker and transfer to the column reservoir, washing the beaker three times with a few ml of de-ionized water. Allow the solution to run through the column at a flow rate of 12 to 14 ml per minute and collect the eluate. Wash the column with two 30-ml portions and with one 50-ml portion of de-ionized water. The column can be used for three successive determinations before regeneration with three times its volume of dilute hydrochloric acid. Titrate the combined eluate and washings (about 150 ml) with 0.20 M sodium hydroxide, using 0.1 ml of phenolphthalein TS as indicator. Calculate the content of C6H5Na3O7.2H2O in g per litre from the following expressions:

1.961n – 1.257P – 1.40C

1.961n – 1.257P – 1.53C

where n = number of ml of 0.20 M sodium hydroxide used in the titration,

P = content of sodium dihydrogenphosphate dihydrate in g per litre determined as prescribed above,

C = content of citric acid monohydrate in g per litre determined as prescribed above, and

C’ = content of anhydrous citric acid in g per litre determined as prescribed above.

FOR REDUCING SUGARS Carry out the tests forreducing sugar (for formula A) described under Anticoagulant Citrate Dextrose Solution, p. 183.

MONOGRAPHS • ANTICOAGULANT AND PRESERVATIVE SOLUTIONS FOR HUMAN BLOOD
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หมายเหตุ / Note : TP II 2011 PAGE 183-185