Analyst, May, 1978, Vol. 103, $9. 509-512 509 Ana lytica I Met hods Corn m i ttee REPORT PREPARED BY THE MEDICINAL ADDITIVES IN ANIMAL FEEDS SUB- COMMITTEE "A" Spectrophotometric Determination of Ronidazole in Animal Feeds Keywords : Ronidazole determination ; animal feeds ; spectrophotometry The Analytical Methods Committee has received and approved for publication the following Report from its Medicinal Additives in Animal Feeds Sub-committee "A." Report The constitution of the Sub-committee responsible for the preparation of this Report was : Mr. J. Markland (Chairman), Mr. R. J. Anderson, Mr. A. G. Croft, Mr. C. E. Dodd, Mr. R. Fawcett, Dr. K. Field, Mr. R. S. Hatfull, Mr. G. E. Kitson, Mr. D. H. Mitchell and Mr. J. A. Stubbles, with Mr. P. W. Shallis as Secretary. Introduction Ronidazole [ (l-methyl-5-nitroimidazol-2-yl)methyl carbamate] is used in poultry feeds for the control and treatment of blackhead; it is also used in pig feeds for the treatment and control of dysentery and as a growth-promoting agent.The normal level of inclusion of the drug in a feed is between 60 and 120 mg k g l . Experimental and Results The most promising method available when the Sub-committee began its work was that proposed by Szalkowski and Kan0ra.l In this method, the nitro group of the drug is split off by alkaline hydrolysis, which is followed by diazotisation of 4-aminobenzoic acid by the liberated nitrite ion and then coupling with N-2-aminoethyl-1-naphthylamine to form a coloured complex. The absorption of the coloured complex is measured at 550nm in a 10-mm cell.Preliminary work on the method by some members of the Sub-committee indicated that it was not specific for ronidazole as other drugs that contain a nitro group, such as dimetrid- azole and nitrofurazone, interfered. There was also some evidence to suggest that interference was encountered when grass meal or fish meal was present in the sample. It was realised that a gas - liquid chromatographic method might prove to be the best for the determination of ronidazole. However, in the absence of any suitable method a t that time, the Sub-committee decided to standardise the conditions of the spectrophotometric method. Subsequently, a gas - liquid chromatographic method based on the formation of a volatile silyl derivative of ronidazole in which no interference from the presence of grass meal, fish meal or other drugs in the feed was observed has been proposed by Harris et aZ.2 The conditions of the method proposed by Szalkowski and Kanoral were examined in detail by members of the Sub-committee.It was found that in common with most other methods of this type, the batch and grade of aluminium oxide used appeared to have a profound effect on the recovery of the drug and in this instance the type and grade of floridin earth used also had an effect. Acceptable batches and types of both materials are available and it is suggested that the suitability for the purpose of the aluminium oxide and the floridin earth should first be checked by taking a standard through the procedure; a recovery of at least 90% should be obtained. In their preliminary work, some members had found that recoveries of ronidazole added to animal feeds were good, despite variable and sometimes large feed blank values.The method makes use of liberated nitrite ion to diazotise 4-aminobenzoic acid and therefore any adventitious contamination with nitrite after cleavage of the molecule will give rise to an apparent ronidazole content. As a result of the exploratory work carried out by510 ANALYTICAL METHODS COMMITTEE : SPECTROPHOTOMETRIC Analyst, Vd. 103 members, a few modifications were incorporated into the published method. A collaborative test was then carried out by applying the modified method to the determination of ronidazole added within each laboratory to a circulated sample of feedingstuff at a level of 60 mg kg-1; the results are shown in Table I.TABLE I DETERMINATION OF RONIDAZOLE IN AN ANIMAL FEED BY THE RECOMMElNDED METHOD Ronidazole added/ Laboratory mg kg-l A 60 60 60 B 60 60 C 60 60 60 D 60 60 60 E 60 60 F 60 60 G 64.8 64.8 H 60 60 60 60 60 Ronidazole found/ mg kg-l 63.6, 64.2 52.8, 54.0 58.8, 57.6 56.3 55.1 51.5 51.6 51.0 60.5 55.8 67.6 63.0 55.0 69.0 69.0 67.3 75.7 61.0 68.0 60.0 59.0 57.0 Recovery, 106.0, 107.0 88.0, 90.0 98.0, 96.0 % 93.8 91.8 85.8 86.0 85.0 loo.s 93.0 96.0 105.0 91.7 115.0 115.0 103.9 116.8 101.7 113.3 100.0 98.3 95.0 Feed blank/ mg kg-l 7.9 7.9 5.2 2.5 2.5 4.4 6.4 3.6 4.5 4.5 7.0 7.0 Corrected recovery, % 80.7 78.7 77.2 81.8 80.8 93.5 82.3 90.0 107.5 107.5 83.1 106.0 Recommendation The Sub-committee recommends that the rnethod given in the Appendix should be used for the determination of ronidazole in animal feeds known to be free from grass meal, fish meal and interfering nitro compounds. APPENDIX Recommended Method for the Determination of Ronidazole in Feeds Scope and Field of Application meal, fish meal and other drugs containing a nitro group that will interfere.The method is for the determination of ronidazole in animal feeds in the absence of grass Principle Ronidazole is extracted from the feed with hot methanol and the extract is cleaned up on a column of aluminium oxide and floridin earth. The extracted drug is hydrolysed with alkali in the presence of copper and the nitrite formed is used to diazotise 4-aminobenzoic acid, which is then coupled with N-2-aminoeth;yl-l-naphthylamine to form a purple complex.The absorbance of the complex is measured at 550 nm. Reagents Methanol.M a y , 1978 DETERMINATION OF RONIDAZOLE I N ANIMAL FEEDS 511 Butan- 1-01. Sodium chloride. Aluminium oxide. Floridin earth. See Note 1. Copper(11) sul@hate solution. Alkaline co$@er solution. For chromatography (see Note 1). Dissolve 2.5 g of anhydrous copper(I1) sulphate in sufficient Add 1.0 ml of the copper(I1) sulphate solution to 100 ml of a Prepare this solution Dissolve 400 mg of 4-aminobenzoic acid in 250 ml of water, Dilute to 500ml with water and Dissolve 50 mg of N-2-aminoethyl-l- Dissolve 30.0 mg of pure ronidazole in sufficient methanol This solution is stable for 1 month if stored in a tightly water to produce 100 ml of solution.5% m/V aqueous solution of sodium hydroxide and mix thoroughly. freshly before use. add 100ml of hydrochloric acid (sp. gr. 1.18) and mix. mix thoroughly. N-2-aminoethyl-1-naphthylamine reagent solution. naphthylamine hydrochloride in 50 ml of water. Ronidaxole stock solutions. to produce 100ml of solution. stoppered container and protected from light, 4-Aminobenxoic acid solution. Cool the solution in an ice - water bath before use. Prepare this solution freshly each day. (A). 1 ml of solution = 300 pg of ronidazole. (E). Transfer 4.00 ml of solution A into a 100-ml calibrated flask, dilute to the mark with This solution is stable for 1 week if stored in a tightly stoppered water and mix thoroughly. container and protected from light. 1 ml of solution zz 12 pg of ronidazole.Ronidaxole working standard solution. Transfer 10.0 ml of solution B into a 100-ml Prepare this solution calibrated flask, dilute to the mark with water and mix thoroughly. freshly each day. Preparation of Chromatographic Column Into the bottom of a glass tube about 400 mm long and 11 mm i.d., constricted at its bottom end to a diameter of 4-5mm, place a small plug of Pyrex glass-wool. Place 3 . 0 g of aluminium oxide into the column and assist settling by gently tamping it with a glass rod. On top of the aluminium oxide place 2.0g of the floridin earth and also tamp this lightly with a glass rod. Procedure Weigh accurately sufficient of the finely divided sample to contain about 600 pg of ronidazole and transfer it into a 250-ml flat-bottomed flask.Add 100.0ml of methanol, place a magnetic stirrer bar in the flask and attach the flask to a reflux condenser. Heat, with stirring, on a magnetic-stirrer hot-plate to maintain a gentle reflux for 30min and then immerse the flask with the condenser still attached in cold water until it reaches room temperature. Transfer 5.0 ml of the clear extract to the top of the chromatographic column and allow it to drain through under gravity. Wash the column with three 5-ml portions of methanol and collect the washings in the same tube. Add 0.1 ml of glacial acetic acid, place the tube in a water-bath a t 50 "C and evaporate to dryness by passing over the surface of the liquid a stream of compressed air carefully controlled to avoid splashing (see Note 2).Dissolve the residue in 10.0 ml of chloroform, add 25.0ml of water, stopper the tube, shake it for 5 min on a mechanical shaker and then centrifuge for about 5 min. Transfer two 10.0-ml portions of the aqueous (top) layer into separate 50-ml centrifuge tubes (A, and A,). To further separate 50-ml centrifuge tubes transfer two 10.0-ml portions of ronidazole working standard solution (B, and B,) and two 10.0-ml portions of water (C, and To the tubes A,, B, and C , add 5.0ml of water and mix. To the tubes A,, B, and C, Transfer the mixture to centrifuge tubes and centrifuge for about 5 min. Collect the eluate in a 50-ml centrifuge tube. C2) -51 2 ANALYTICAL ME.THODS COMMITTEE add 5.0 ml of alkaline copper solution arid mix thoroughly. Place all tubes (without stoppers) into a stirred water bath at 80 "C, allow 1-2 min for the tubes and their contents to become warm and then insert the stoppers tightly.After 1 h, remove the tubes from the water-bath, momentarily loosen and replace the stoppers and then immerse the tubes in an ice - water bath until the temperature has been reduced to 3-5 "C (about 5 min). Remove the tubes from the ice - water bath, add 5.0 ml of cold 4-aminobenzoic acid solution to the contents of each tube and set them aside for 2 min. Then add 1.0 ml of N-Z-amino- ethyl-l-naphthylamine reagent solution to the contents of each tube, mix and set them aside at room temperature for 20 min. Finally, add 5.0 g of sodium chloride and 5.0 ml of butan-1-01 to the contents of each tube, insert the stoppers and shake each tube vigorously for 2-3 min.Allow the layers to separate and with a pipette carefully transfer the butanol layer to a small (about 15-ml) centrifuge tube. Spin the tubes in a centrifuge at about 2 000 rev min-l for 3 min. Measure the absorbances of the clarified butanol solutions from each of the tubes in 10-mm cells against butan-1-01. Calculation of Results Calculate the concentration of ronidazole in the feed sample from the expression 2 x 100 x x 2 x W x Y Ronidazole/mg kg-l = where X = corrected absorbance of sample, ie., (A, - A,) - (C, - C,). Y = corrected absorbance of standard, Le., (B, - B,) - (C, - C2). 2 = mass of ronidazole (micrograms) in ithe standard (usually 12 pg). W = mass of feed (grams) taken for analysis. NOTES- Various makes and grades of aluminium oxide, both neutral and basic, and of floridin earth have been found to be satisfactory. Before use a standard should be put through the column, which can be considered to be satisfactory for use if a recovery of at least 90% is obtained. It is essential to remove impurities from the compressed air supply used at the evaporation stage. For this purpose, pass the air through a train of three gas-washing bottles, the first containing a saturated solution of potassium hydroxide, the second conctmtrated sulphuric acid saturated with chromium(II1) oxide and the third Pyrex glass-wool. 1. 2. References 1. 2. Szalkowski, C. R., and Kanora, J., J . Ass. 08. Analyt. Chem., 1969, 52, 101. Harris, J. R., Baker, P. G., and Alliston, G., A4naZyst, 1977, 102, 580.