Appendix XV D. Free Formaldehyde

(Ph. Eur. method 2.4.18)

Use method A, unless otherwise prescribed. Method B is suitable for vaccines where sodium metabisulfite has been used to neutralise excess formaldehyde.

METHOD A

For vaccines for human use, prepare a 1 in 10 dilution of the vaccine to be examined. For bacterial toxoids for veterinary use, prepare a 1 in 25 dilution of the vaccine to be examined.

To 1 mL of the dilution, add 4 mL of water R and 5 mL of acetylacetone reagent R1. Place the tube in a water-bath at 40 °C for 40 min. Examine the tubes down their vertical axes. The solution is not more intensely coloured than a standard, prepared at the same time and in the same manner, using 1 mL of a dilution of formaldehyde solution R containing 20 µg of formaldehyde (CH₂O) per millilitre, instead of the dilution of the vaccine to be examined.

METHOD B

Test solution Prepare a 1 in 200 dilution of the vaccine to be examined with water R. If the vaccine is an emulsion, prepare an equivalent dilution using the aqueous phase separated by a suitable procedure (see below). If one of the methods described below is used for separation of the aqueous phase, a 1 in 20 dilution of the latter is used.

Reference solutions Prepare solutions containing 0.25 g/L, 0.50 g/L, 1.00 g/L and 2.00 g/L of CH₂O by dilution of formaldehyde solution R with water R. Prepare a 1 in 200 dilution of each solution with water R.

To 0.5 mL of the test solution and of each of the reference solutions in test-tubes, add 5.0 mL of a freshly prepared 0.5 g/L solution of methylbenzothiazolone hydrazone hydrochloride R. Close the tubes, shake and allow to stand for 60 min. Add 1 mL of ferric chloride-sulfamic acid reagent R and allow to stand for 15 min. Measure the absorbance (2.2.25) of the solutions at 628 nm. Calculate the content of formaldehyde in the vaccine to be examined from the calibration curve established using the reference solutions. The test is invalid if the correlation coefficient (r) of the calibration curve is less than 0.97.

Emulsions If the vaccine to be examined is an emulsion, the aqueous phase is separated using a suitable procedure and used for preparation of the test solution. The following procedures have been found suitable.

(a) Add 1.0 mL of the vaccine to be examined to 1.0 mL of isopropyl myristate R and mix. Add 1.3 mL of 1 M hydrochloric acid, 2.0 mL of chloroform R and 2.7 mL of a 9 g/L solution of sodium chloride R. Mix thoroughly. Centrifuge at 15 000 g for 60 min. Transfer the aqueous phase to a 10 mL volumetric flask and dilute to volume with water R. If this procedure fails to separate the aqueous phase, add 100 g/L of polysorbate 20 R to the sodium chloride solution and repeat the procedure but centrifuge at 22 500 g.

(b) Add 1.0 mL of the vaccine to be examined to 1.0 mL of a 100 g/L solution of sodium chloride R and mix. Centrifuge at 1000 g for 15 min. Transfer the aqueous phase to a 10 mL volumetric flask and dilute to volume with water R.

(c) Add 1.0 mL of the vaccine to be examined to 2.0 mL of a 100 g/L solution of sodium chloride R and 3.0 mL of chloroform R and mix. Centrifuge at 1000 g for 5 min. Transfer the aqueous phase to a 10 mL volumetric flask and dilute to volume with water R.