สารบัญ

Category Anticoagulant for storage of whole blood.

         Anticoagulant Citrate Phosphate Dextrose Adenine Solution is a sterile solution of Citric Acid, Sodium Citrate, Sodium Dihydrogenphosphate Monohydrate, Dextrose, and Adenine in Water for Injection. It contains, in each 1000 ml, not less than 2.11 g and not more than 2.33 g of sodium dihydrogenphosphate monohydrate (NaH₂PO₄.H₂O); not less than 30.30 g and not more than 33.50 g of dextrose (C₆H₁₂O₆.H₅O); not less than 19.16 g and not more than 21.18 g of total citrate, expressed as citric acid, anhydrous (C₆H₈O₇); not less than 6.21 g and not more than 6.86 g of sodium (Na); and not less than 0.247 g and not more than 0.303 g of adenine (C₅H₅N₅). It contains no antimicrobial agent.

Citrate Phosphate Dextrose Adenine Solution (CPDA-1)

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

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

See also under Anticoagulant Citrate Phosphate Dextrose Solution, p. 183.

pH 5.0 to 6.0 (Appendix 4.11).

Chloride Not more than 0.0035 per cent w/v (Appendix 5.2), a 10-ml portion shows no more chloride than that corresponds to 0.50 ml of 0.020 M hydrochloric acid.

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

Assay

         FOR TOTAL CITRATE AND TOTAL PHOSPHATE

         Mobile phase Transfer an appropriate volume of water (resistivity not less than 18 megohm-cm) to a suitable container, and degas with helium for not less than 20 minutes. Add an appropriate volume of a 50 per cent w/w carbonate-free sodium hydroxide or potassium hydroxide to obtain a 20 mM. Alternatively, a 20 mM sodium hydroxide or potassium hydroxide eluent can be generated electrolytically using an automatic eluent generator. (Note Protect Mobile phase from atmospheric carbon dioxide.)

         Standard preparation Dissolve Citric Acid RS and sodium dihydrogenphosphate in freshly prepared 1 mM sodium hydroxide to obtain a solution having known concentrations of about 20 μg per ml and 12 μg per ml of citrate and phosphate (PO₄), respectively.

         Assay preparation for total citrate assay Pipette 10 ml of Anticoagulant Citrate Phosphate Dextrose Adenine Solution into a suitable volumetric flask and add freshly prepared 1 mM sodium hydroxide to obtain a solution containing about 20 μg per ml of citrate in 1 mM sodium hydroxide.

         Assay preparation for total phosphate assay Pipette 5 ml of Anticoagulant Citrate Phosphate Dextrose Adenine Solution into a suitable volumetric flask and add freshly prepared 1 mM sodium hydroxide to obtain a solution containing about 12 μg per ml of phosphate in 1 mM sodium hydroxide. Chromatographic systems The chromatographic procedure may be carried out using (a) a guard column (5 cm × 4 mm) and a stainless steel analytical column (25 cm × 4 mm), both packed with a hydroxide selective strong anion-exchange resin consisting of a highly crosslinked core of 13-μm microporous particles having a pore size less than 10 Å and consisting of ethylvinylbenzene cross-linked with 55 per cent divinylbenzene with a latex coating composed of 85-nm diameter microbeads bonded with alkanol quaternary ammonium ions (6 per cent), (b) Mobile phase at a flow rate of about 2 ml per minute, (c) an electrochemical detector with suppressed conductivity detection using either a micromembrane anion autosuppressor or a suitable chemical suppression system. All columns are maintained at a temperature of 30º. (Note An anion trap column designed to remove trace anion contaminants in the Mobile phase should be added to the column assembly before the injector.)

         To determine the suitability of the chromatographic system, chromatograph Standard preparation, and record the peak area responses as directed for Procedure: the symmetry factor is not more than 2.0, and the relative standard deviation of the peak areas for citrate and phosphate, for six replicate injections of StandardPreparation, is not more than 1.5 per cent.

         Procedure Separately inject equal volumes (about 10 μl) of Standard preparation and Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas for citrate and phosphate, as appropriate.

         Calculation Calculate the quantity, in mg, of anhydrous citric acid (C₆H₈O₇) in the volume of the Solution taken by the expression:

0.001(192.12/189.10) C_SD(r_U/r_S),

in which 192.12 is the molecular weight of anhydrous citric acid; 189.10 is the molecular weight of citrate (C₆H₅O₇); CS is the concentration, in μg per ml, of citrate in Standard preparation; D is the dilution factor; and rU and rS are the citrate peak areas obtained from Assay preparation for total citrate assay and Standard preparation, respectively.

         Calculate the quantity, in mg, of phosphate, expressed as sodium dihydrogenphosphate monohydrate (NaH₂PO₄.H₂O), in the volume of the Solution taken by the expression:

0.001(137.99/94.97)C_SD(r_U/r_S),

in which 137.99 is the molecular weight of sodium dihydrogenphosphate monohydrate; 94.97 is the molecular weight of phosphate (PO₄); C_S is the concentration, in μg per ml, of phosphate in Standard preparation; D is the dilution factor; and r_U and r_S are the phosphate peak areas obtained from Assay preparation for total phosphate assay and Standard preparation, respectively.

FOR SODIUM

         Lithium diluent solution Transfer 1.04 g of lithium nitrate to a 1000-ml volumetric flask, add a suitable nonionic surfactant, then add water to volume, and mix. This solution contains 15 mEq of lithium per litre.

         Standard preparation Transfer 8.18 g of sodium chloride, previously dried at 105º for 2 hours and accurately weighed, to a 1000-ml volumetric flask, dilute with water to volume, and mix. This solution contains 140 mEq of sodium per litre. Transfer 50 μl of this solution to a 10-ml volumetric flask, dilute with Lithium diluent solution to volume, and mix.

         Assay preparation Pipette 25 ml of Anticoagulant Citrate Phosphate Dextrose Adenine Solution into a 50-ml volumetric flask, dilute with water to volume, and mix. Transfer 50 μl of this solution to a 10-ml volumetric flask, dilute with Lithium diluent solution to volume, and mix.

         Procedure Concomitantly measure the absorbances of Standard preparation and Assay preparation at the maximum at about 589 nm, using Lithium diluent solution as the blank (Appendix 2.2).

         Calculation Calculate the quantity, in g, of Na in 1000 ml of the Solution taken by the expression:

2(8.18)(22.99/58.44)(r_U/r_S),

in which 8.18 is the weight, in g, of sodium chloride taken to make Standard preparation; 22.99 is the atomic weight of sodium; 58.44 is the molecular weight of sodium chloride; and r_U and r_S are the sodium emission readings obtained from Assay preparation and Standard preparation, respectively.

         FOR DEXTROSE Tare a clean, medium-porosity filtering crucible containing several carborundum boiling chips or glass beads. Pipette 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 Anticoagulant Citrate Phosphate Dextrose Adenine 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 ethanol. Dry the crucible and contents at 110º to constant weight. Perform a blank determination, and make any necessary correction. Each mg of cuprous oxide precipitate obtained is equivalent to 0.496 mg of C₆H₁₂O₆.H₂O.

FOR ADENINE

         Mobile phase Dissolve 3.45 g of ammonium dihydrogenphosphate in 950 ml of water in a 1000-ml volumetric flask, add 10 ml of glacial acetic acid, dilute with water to volume, mix, pass through a membrane filter having a 1-μm or finer porosity, and degas.

         Standard preparation Dissolve accurately weighed quantities of Adenine RS in diluted hydrochloric acid (1 in 120) in three separate volumetric flasks, dilute with the diluted hydrochloric acid solution to volume, and mix to obtain Standard preparations having known concentrations of about 250, 275, and 300 μg of adenine per ml, respectively. Protect from light.

         Resolution solution Prepare a solution containing Adenine RS and purine, each at about 275 μg per ml, in diluted hydrochloric acid (1 in 120).

         Chromatographic system The chromatographic procedure may be carried out using (a) a stainless steel column (30 cm × 4 mm) packed with irregular or spherical, totally porous silica gel having a chemically bonded, strongly acidic cation-exchange coating, (3 to 10 μm), (b) Mobile phase at a flow rate of about 2.0 ml per minute, and (c) an ultraviolet photometer set at 254 nm.

         To determine the suitability of the chromatographic system, chromatograph not less than four injections (about 20 μl) of Resolution solution: the relative standard deviation of the peak response of adenine is not more than 2.5 per cent, the relative standard deviation of the retention time of adenine is not more than 2.0 per cent, and the resolution factor of adenine and purine is not less than 3.0.

         Procedure Separately inject equal volumes (about 20 μl) of Anticoagulant Citrate Phosphate Dextrose Adenine Solution and Standard preparation, record the chromatograms, and measure the responses for the major peaks. Plot the responses against the concentrations, in mg, of Adenine RS per ml of Standard preparations.

         Calculation Calculate the quantity, in mg, of C₅H₅N₅ in each ml of the Solution taken as the value read directly from the Standard curve corresponding to the response obtained from the portion of the Solution chromatographed.