Determination of Furazolidone and Nitrofurazone in Chicken Tissues RONALD J. HERRETT and JAMES A. BUZARD Biochemistry Section, Eaton laboratories, The Norwich Pharmacal Co., Norwich, N. Y.
F A method for the determination of furazolidone and nitrofurazone in chicken tissues is based on the formation and colorimetric estimation of 5nitro-2-furaldehyde phenylhydrazone. A chromatographic separation of the phenylhydrazone from nonspecific liver chromogens permits visual demonstration of the nitrofuran in concentrations as low as 0.5 p.p.m. and permits quantitative determination of these nitrofurans in chicken liver at 1 to 5 p.p.m. with an accuracy of 92 to 104%. A nonchromatographic method is used for fat and muscle residue analysis. The recoveries for these tissues ranged from 97 to 1 0 8 ~ o at 1 and 5 p.p.m.
T-
use of furazolidone, 3 (5 - nitrofurfurylideneamino) - 2oxazolidinone (Furoxone), and nitrofurazone, 5-nitro-Zfuraldehyde semicarbazone (Furacin), (Structures 1 and 2) in the treatment and control of various poultry diseases (5, 7) has emHE INCREASING
Structure 1. Nitrofurazone, 5-nitro-2-furaldehyde semicarbazone
phasized the need for an analytical method to determine quantitatively low concentrations of these drugs in tissues. These nitrofurans can be determined in plasma and poultry feeds at low concentrations by a method based on the formation and colorimetric estimation of Cnitro-Zfuraldehyde phenylhydrazone (3, 4). This method has been diflicult to apply to highly pigmented tissues such as liver because of nonspecific colorimetric interference. Because of this, relatively little published information on the concentration of these nitrofurans in the tissues of treated poultry has been available ( I , 6). The phenylhydrazone colorimetric method has been modified to overcome these daculties. Column chromatography has been employed to separate the phenylhydrazont- from most of the extraneous materiais prior to the spectrophotometric mclasurement.
1676
0
ANALYTlCAL CHEMISTRY
Methods of satisfactory sensitivity, accuracy, and precision have been developed for the estimation in chicken muscle, liver, and fat. MATERIALS AND METHODS
Reagents and Instrument. The reagents used include phenylhydrazine hydrochloride 1.5% in water, prepared fresh daily and refrigerated when not in use; N,N-dimethyl-
Structure 2. Furazolidone, 3-(5-nitrofurfurylideneamino)- 2 oxazolidinone
-
formamide (b.p. 150" t o 154" C.); ethyl acetate, anhydrous; crystalline standards of the nitrofurans (Eaton Laboratories). A Beckman Model DU spectrophotometer was used throughout. Preparation of Standard. Dissolve an accurately weighed sample of about 50 mg. of the crystalline standard nitrofuran in 50 ml. of N,.Vdimethylformamide in a 100-ml. volumetric flask and make u p to volume with distilled water. Dilute 5 ml. of this solution t o 50 ml. with water. Dilute 5 ml. of the aqueous dilution just made t o 50 ml. with water to obtain a solution containing 5 pg. of the nitrofuran per ml. Protect dilute nitrofuran solutions f r o m direct sunlight or fluorescent lighting at all times to avoid decomposition. Prepare standards daily. Procedure for Liver. Livers excised from sacrificed birds are quickly frozen in powdered dry ice and stored in a freezer until time of analysis. The livers are then thawed in a refrigerator and 5 grams of each cold liver are weighed into a separate Potter-Elvehjem type, glass tissue grinder containing 8 ml. of cold 5% metaphosphoric arid. The samples are then homogenized and the homogenate3 are quantitatively transferred into 50-ml. p a d u ated centrifugp tubes with 3 nil. of cold 57" metaphosphoric acid and cold witer t,o bring the volume to 30 nil. T o each tube is added 3 nil. of l.5GG phenylhydrazine hydrochloride. Standart-la of t,he appropriate nitrofuran we analJ-zed
simultaneously with the liver samples. One, 2, and 5 ml. of the 5 pg. per ml. of solution are placed in separate 50-ml. graduated centrifuge tubes containing 11 ml. of cold 5% metaphosphoric acid, 3 ml. of 1.5y0phenylhydrazine hydrochloride, and enough water to make the final volume 33 nil. The samples and standards must be kept cold during the entire process. The liver homogenates and standards are then heated in a water bath a t 70" C. for 10 minutes and cooled. To each tube is added 3 ml. of 12N hydrochloric acid. The contents are mixed bx shaking and heating 25 minutes a t 70 C., cooled, and transferred to 50-ml. glass-stoppered centrifLge tubes. The contents are shaken with 10 ml. of toluene for approximately 1 minute, centrifuged, and 8 ml. of the toluene layer is chromatographed on an aluminum oxide column as follows: Chromatographic columns, 25 X 1 cm., packed dry with anhydrous alumina [reagent aluminum oxide, XIerck, suitable for chromatographic adsorption, (Cat. No. 71707)] to a height of 6 to 7 cm. on a glass wool plug, are washed with reagent grade toluene and a second glass wool plug is inserted a t the top. Before the addition of the samples, the columns are equilibrated with toluene. After the addition of the 8-ml. sample, 10 ml. of toluene is passed through the column. At this point all the colored material, including the phenylhydrazone, is retained a t the top of the adsorbent. Upon the addition of tolueneethyl acetate (3 to I), t'he liver sample containing the nitrofuran separates into four bands. From the fastest to the slowest moving bands, these substances are as follows: a yellow liver material, the red phenylhydrazone derivative, a second yellow liver material, and finally, a stationary brown band. Sitrofuran standards yield only the red phenylhydrazone band. This solvent system 1s used to elute the faster yellow material s1.d to separate the red band from the second yellow material. -4fter the first rellow band has been eluted, a 1 to 1 mixture of toluene and ethyl acetate is added until the red band is near the bottom of the column. When the red band approaches the bottom of the column, pure ethyl acetate is used and the phenylhydrazone is eluted from volume of 10 to 1.5 ml. the column. -i of eluate was necessary to remove all the prienylhydrazone from the column withoiit contamination with the second yellow band. The eluate containing t,he phenyl-
hydrazone is concentrated to less than 0.5 ml. with a Rinco flash evaporator (about 15 minutes a t room temperature on a water aspirator pump) and made to 5 ml. with toluene and the absorbance determined from 400 to 460 mp against a toluene blank. A standard curve for each nitrofuran is constructed by plotting the absorbances a t 440 mp of the three concentrations of the nitrofuran used, against the amount of that nitrofuran originally present (5, 10, and 25 fig. per tube). The absorbance of the control liver sample (from a nonmedicated bird) is subtracted from that of the liver sample from the medicated bird and the amount of nitrofuran present in the sample is read directly from the standard curve. The nitrofuran concentration in the original liver sample, in parts per million, is obtained by dividing the micrograms found per tube by 5, the weight in grams of the initial liver sample. Criteria for positive identification of the nitrofuran in the liver are visual identification of the red band on the chromatographic column plus a difference curve of the eluate (absorbance us. wave length of treated minus that of untreated samples) showing an absorption maximum at 440 mp (Figure 1). EXPERIMENTAL
Characteristics of the Color Produced. Toluene extracts of 5-nitro2-furaldehyde phenylhydrazone prepared from furazolidone and nitrofurazone display an absorption peak at 440 mp (Figure 1). The stability of the color produced and the specificity of the reaction have been demonstrated in earlier papers (3, 4). Extraction of the Nitrofuran from Liver. Extraction of the nitrofuran from liver has been a problem since nitrofurans are known to undergo degradation rapidly in the presence of liver nnd other tissues ( 2 ) . Inactivation of the liver by metaphosphoric acid mid heating proved completely satisfactory for quantitatively extracting the nitrofuran from liver. This is demonstrated in tissue recovery experiments described below. Stability of Furazolidone and Nitrofurazone in Acid Solution. The use of metaphosphoric acid caused some difficulty in early recovery experiments with nitrofurazone. Both nitrofurazone and furazolidone are susceptible to acid hydrolysis; the former is 2 to 3 times as susceptible as the latter (6). While the concentration of furazolidone standards was not altered by standing in 2% metaphosphoric acid a t room temperature for several hours, a considerable loss was noted with nitrofurazone standards. This loss was of consequeoce since the desired intermediate, 5-nitro-2-furaldehyde, is unstable under these conditions and must be trapped its it is formed.
I
I
W A V E LENGTH MY
Figure 1. A. 6.
C.
Absorption spectra
Control-corrected difference spectrum derived from l p.p.m. of furazolidone or nitrofurazone added to chicken liver 5-Nitro-2-furaldehyde phenylhydrazone in toluene, equivalent to 1 p.p.m. of furazolidone or nitrofurazone in original standard solution Unrnedicated control chicken liver
I n preliminary experiments, low absorbances for nitrofurazone standards were obtained if the standards were allowed to stand a t room temperature for several hours in l.8yOmetaphosphoric acid. This concentration of acid did not have any detectable effect on the nitrofurazone in the liver samples because the effective acid concentration was reduced by the precipitation of the liver protein plus the fact that the lower temperatures employed with the liver samples minimized the rate of hydrolysis. 'I5: difficulties encountered with wtaphosphoric acid in standard nitrofwazone solutions were overcome by keeping the standards cold and by adding the phenylhydrazine reagent
Table 1.
Recovery of Furazolidone and Nitrofurazone from Chicken Tissues
Tissue Liver
Compound Furazolidone Nitrofurazone
Muscle
Furazolidone Nitrofurazone
Fat
to the standards prior to the addition of nitrofurazone. Chromatographic Separation of Phenylhydrazone Derivative. T o detect small quantities of nitrofuran in liver samples by the phenylhydrazone method, i t was necessary to reduce to a minimum the absorbance of the toluene extract due to large amounts of interfering materials extracted from the liver. This was accomplished by column chromatography on aluminum oxide. The separation and elution of the phenylhydrazone derivative are accomplished as described in the procedure for liver. Apparent drug concentration of chromatographed, untreated liver samples is of the order of 0.6 p.p.m., that for nonchromatographed liver samples exceeds 10 p.p.m. The appearance of the red band on the column serves as a qualitative test for the positive identification of nitrofuran. A minimum of 2 pg. of &nitro2-furaldehyde phenylhydrazone can be detected and the movement followed down the column. This amount corresponds to 0.5 p.p.m. of nitrofuran in the liver. Absorbance of Eluates. Standard curves of the phenylhydrazone prepared from the nitrofurans follow Beer's law in the range zero t o 25 pg. of the nitrofuran concerned per tube. Different slopes are obtained due to molecular weight differences, the extent of conversion of the various derivatives, and the effect of slight variation in experimental conditions (4). It is, therefore, necessary to run standard solutions of the nitrofuran under investigation simultaneously with each tissue analysis. Under these conditions, quantitative recoveries were obtained, as described below. Procedure for Fat and Muscle. Frozen samples of muscle or fat are thawed in the cold and 5 grams of minced cold tissue is added to 10 ml. of hot (90' t o 100" C.) 50% N , N -
Furazolidone Nitrofurazone
pg./Gram Tissue
Added 1.00 2.00 5.00 1 .OO
2.00 5.00 1 .oo 5.00 1 .OO 5.00 1.00 5.00
%
Found 1.00 2.07 5.03 0.92 1.87 4.79 0.97 4.91 1.04 5.40
Recovered 100.0 103.5 100.6 92.0 93.5 95.8 97.0 98.2 104.0 108.0
1.06
106.0
4.85
97.0 99.0 104.0
1 .oo
0.99
5.00
5.17
VOL. 32, NO. 12, NOVEMBER 1960
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dimethyiformamide (DMF) in 50ml. graduated centrifuge tubes. The tubes are heated in a boiling water bath for 30 minutes. T h e volume is brought t o 15 ml. with distilled water. The entire contents of the fat samples are extracted with 12 ml. of toluene and the toluene extract is discarded. For color development, 10 ml. of the aqueous DMF extract is added to 1 ml. each of 1.5% phenylhydrazine hydrochloride and 12N hydrochloric acid. This mixtiire is heated 25 minutes at 70" C., cooled, and extracted with 5 ml. of toluene. The absorbance of the toluene layer is determined with the spectrophotometer from 400 to 460 mp using pure toluene as a blank. Aqueous standards of nitrofuran containing 5, 10, and 25 pg. per 10 ml. are treated in exactly the same manner as described for f a t and muscle. A standard curve is constructed in the usual manner and
the control-corrected absorbance is used to calculate the nitrofuran content of the tissue. The difference curve must show a maximum of 440 mp t o prove the presence of the nitrofuran. Tissue Recovery Experiments. The
accuracy and precision of this method were demonstrated by carrying out tissue recovery experiments. I n these experiments, known amounts of nitrofurazone or furazolidone were added to the tissue prior to extraction. Typical results of these experiments are presented in Table I. With fat and muscle the background absorbance of the control tissue was low enough (equivalent t o less than 1 p.p.m.) so that chromatographic separation of the phenylhydrazone derivative was not required.
LITERATURE CITED
(1) Belloff, G . B., Buzard, J. A., Roberts, H. D. B., Poultry Sci. 37,223 (1958). (2) Bender, R. C., Paul, H. E., J. Biol. Chem. 191, 217 (1951).
(3) Buzard, J. A., Ells, V. R., Paul, M. F., J. Assoc. O&. Agr. Chemists 39, 512 (1956). (4) Buiard, J. A., Vrablic, D. M., Paul, M. F., Antibiotics & Chemotherapy 6 , 702 (1956). (5) Maftin, J. E., Mich. State Univ. Vetennarian 19, 95-101, 119 (winter lQ5Q).
(6) Paul, M. F., Paul, H. E., Bender, R. C., Kopko, F., Harrington, C. M., Ells, V. R., Buzard, J. A., Antibiotics &
Chemotherapy, in press. (7) Proc. 2nd Natl. Symposium on Kitrofurans in Agriculture, Univ. of Georgia, Athens, Ga., Mar. 27-28, 1958.
RECEIVED for review February 23, 1960. Accepted June 10, 1960.
Photometric and Visual Titration of Certain Alkaloids in Glacial Acetic Acid Using Malachite Green as Indicator SAMUEL M. TUTHILL, ORLAND W. KOLLING', and KARL H. ROBERTS* Mallinckrodf Chemical Works, St. louis, Mo.
,Certain naturally occurring opium alkaloids, such as codeine, cryptopine, morphine, thebaine, narcotine, and papaverine, may b e assayed by titration with perchloric acid in glacial acetic acid solutions with malachite green as the indicator. The change of color at the end point is more distinct and more nearly coincidental with the potentiometric end point than is that of the often used crystal violet. The end point with malachite green may b e determined visually or spectrophotometrically. The visual procedure is more rapid and can be used for routine work, if desired. The method is particularly advantageous when applied to the weakly basic alkaloids such as papaverine and narcotine, since they cannot b e titrated satisfactorily in aqueous solution.
0
THE MANY METHODS utilizing titration in nonaqueous media which have been developed in recent years, the titration of weak bases with a solution of perchloric acid in glacial acetic acid is probably the most familiar. In such titrations, crystal violet is the indicator most commonly used for the colorimetric determination of the end 1 Present address, Department of Chemiqtry, Southwestern College, lVhbeld, F
Kan
Present address, !vlaGe:in c'o , L'leveland 15, Ohio. 1678
('hvmlc:t'
ANALYTICAL CHEMISTRY
point. The major difficulty in its use is the variety of color changes which it undergoes during a titration ( 5 ) . Seaman and Allen ( 7 ) concluded that a preconceived color change generally cannot be used in the titration of different substances; instead they stated that the correct color change for a given determination must he sciected by obserhlng the color of crystal violet a t the potentiometric end point. The photometric titration of weak acids in aqueous solutions with and without added indicators has been studied by Goddu and Hum(: ( 2 ) , and Reilley and Schwizer (6)have shoivn that certain bases, which have no absorbance in eithcr the acidic or basic form, may be titrated photomctrically in glacial acetic acid by adding a wenkcr base, which does absorb. During a study of the absorption spectra 0: crystal violet and malachite green in glacial acetic acid in the presence of varjjng quantities of perchloric acid, it was observed that as the mole ratio of perchioric acid to crystal violet increased, the atxorption mnsimum of crystal violet a t 5% nw decreased, and an nh.cor:)tion mnsini:im XppPared a t 6?0 m s . Tllc, 1sttc.r mzsimuin decreased in inti,r!sity, .in(! :t tliird z~xirr.cm~:Lt 440 i7.u ; 1 1 ( . 1 c : i d i:l intcnric tr
These observations are similar to those reported by Conant and Werner (1). The corresponding family of absorption spectra for malachite green shoxed that an absorption niaximum'&t 622 mp decreased as the ratio of perchloric acid to the indicator increased and that a maximum a t 450 inp increased at the same time. The sharp changes in absorption of these dyes with an increase in the quantity of added acid suggested their use as indicators for the photometric titration of organic bases in glacial acetic acid. Since 3Iallinckrodt Chcmical Works is engagrd in the commercial extraction of naturally occurring opium alkaloids, the possibility of assaying certain of these alkaloids by photometric titration, using the above-mentioned dyes as indicators was investigated. This paper describcs the development of a procedure for such titrations. Basic impurities, including other alkaloids, interfere in the procedure, since they are titrat,ed Kith the alkaloid being assayed.
