DETERMINATION OF ASCARIDOLE

7. DETERMINATION OF ASCARIDOLE

The analysis of wormseed oils offers some difficulty because of the lack of a satisfactory method for the determination of the active principle, ascaridole.
"The United States Pharmacopoeia"109 based the official determination on the fact that ascaridole is soluble in dilute acetic acid. The technique consists of shaking a measured volume of the oil in a cassia flask with 60 per cent acetic acid, determining the volume of undissolvedoil, and calculating the ascaridole content by difference. This is a method developed by Nelson.110 Although this procedure represents one of the simplest determinations for ascaridole, it suffers from the fact that the analytical values are far from accurate. It has been found that normal oils containing as much as 70 to 80 per cent ascaridole (as indicated by solubility, gravity, distillation, and other methods for the determination of ascaridole) often analyze as low as 55 per cent by the Nelson method. Furthermore, Reindollar111 has shown that "hi-test" oils containing large amounts of ascaridole give results by this method that are too high. A further disadvantage of the Nelson method lies in the fact that the determination is by no means specific for ascaridole; additions of cineole or cineole-containing oils analyze as ascaridole. This is also true of many other oxygenated compounds, such as terpineol.
Procedure: Place 10 cc. of oil wormseed, measured from a pipette, in a 100 cc. cassia flask. Add 50 cc. of a solution of acetic acid made by diluting 60 cc. of glacial acetic acid with distilled water to measure 100 cc. Shake the mixture well for 5 min. Add sufficient of the acetic acid solution to raise the lower limit of the oily layer within the graduated portion of the neck and allow the liquids to separate, rotating the flask from time to time. Note the volume of the oily layer.
Percentage of ascaridole = 10(10 - cc. of unreacted oil)
In order to overcome these difficulties, "The United States Pharmacopoeia"112 later abandoned the Nelson method and then substituted the Cocking and Hymas113 procedure, previously official in "The British Pharmacopoeia."114 The procedure given below is based upon this official method:
Assay: Place about 2.5 g. of oil chenopodium, accurately weighed, in a 50 cc. volumetric flask, fill to the mark with 90% acetic acid, mix well, and transfer a portion of this freshly prepared solution to a burette, graduated in 20ths of a cc. Into a glass-stoppered Krlenmeyer flask measure, from graduated cylinders, 3 cc. of a solution of potassium iodide (prepared by dissolving 8.3 g. of potassium iodide in sufficient distilled water to make 10 cc. of solution), 5 cc. of concentrated hydrochloric acid, and 10 cc. of glacial acetic acid. Immerse the flask in a freezing mixture until the temperature is reduced to 3, add quickly about 5 cc. of the acetic acid solution of the oil, mix it with the cooled reagent as rapidly as possible, and observe the volume drawn from the burette after 2 min. (to allow for draining). Set the stoppered flask aside at a temperature between 5 and 10 for exactly 5 min. ; then, without diluting, titrate the liberated iodine with tenth-normal sodium thiosulfate. At the same time, conduct a blank test, but dilute the reagent with 20 cc. of distilled water before titrating the liberated iodine. The difference between the two titrations represents the iodine liberated by ascaridole. Each cc. of tenth-normal sodium thiosulfate is equivalent to 0.00665 g. of C10H16O2.115
If all conditions as outlined are rigidly followed, this method gives analytical results that are reproducible and relatively accurate for normal American oils. If, however, the ascaridole content is abnormally low, the results will not be sufficiently accurate.
Since this method is based on the oxidation of potassium iodide by the peroxide, ascaridole, and the subsequent determination of the amount of free iodine, additions of oxygenated constituents should not increase the apparent ascaridole content unless the added compound is capable of oxidizing the potassium iodide under the experimental conditions. Furthermore, since the liberated iodine is capable of being absorbed by unsaturates in the oil, it is very important to maintain the low temperatures as indicated in the procedure, to keep this secondary reaction at a minimum.
The tentative method for the determination of ascaridole as outlined in the Methods of Analysis of the Association of Official Agricultural Chemists116 proves too cumbersome for rapid commercial analyses and control. This method, developed by Paget,117 involves the reduction of ascaridole by titanium trichloride. It requires that the solution be protected from atmospheric oxygen ; this entails storage of the solution under an atmosphere of hydrogen, and titrations under an atmosphere of carbon dioxide. According to the experiments of Reindollar,118 the results obtained by this method are fairly concordant with those obtained by the method of Cocking and Hymas. The Association of Official Agricultural Chemists' method is given below, exactly as it appears in this official work :
Procedure: "Weigh 1 mi of the oil in 100 ml volumetric flask and dilute to volume with alcohol. Place 50 ml of the TiCl3 soln in Erlenmeyer flask through which current of CO2 is passing. Fit flask uith Bunsen valve, add 10 ml of diluted soln of the oil, close flask (with the Bunsen valve), and heat contents almost to boiling for 2 min. (Prolonged heating has no effect if contents are not boiled vigorously.) If pale violet color of the TiCl3 disappears, add more reagent to insure excess. (Formation of a white precipitate does not interfere with determination.) Add 1 ml of 5% NH4CNS soln and titrate back excess of TiCl3 with the FeNH4(S04)2 soln in CO2 atmosphere until faint, permanent, brownish red color is obtained.
"Subtract quantity of FeNH4(SO4)2 soln used, expressed in equivalent mg of TiCl3, from number of mg of TiCl3 taken. Difference is number of mg of TiCl3 oxidized by oil taken. Convert mg of TiCl3 oxidized into ascaridole by dividing by factor 1.284 (1 g of ascaridole is reduced by 1.284 g of TiCl3). "Example : 0.9600 g of oil was made up to 100 ml and 10 ml aliquot was heated with 50 ml of the TiCl 3 soln (1 ml containing 0.0034 g of TiCl3). It then required 5.9 ml of the reagent, each ml equivalent to 0.01545 g to TiCl3, to back titrate. Grams of
TiCl3 oxidized is numerically equal to (50 X 0.0034) - (5.9X 0.01545), or 0.07885. Weight of oil in the aliquot was 0.0960 g. Hence percentage of ascaridole =   0.07885x100/0.096x1.284= 72.1%.

a) Standard ferric ammonium sulfate soln. 

Dissolve 39.214 g of pure, crystallized Fe(NH4)2(SO4)2.6H2O in 200 ml of H2O in liter flask, add 30 ml of H2SO4 , and mix well. Weigh exactly 3.16 g of KMnO4, dissolve in 200 ml of warm H2O, and slowly add to soln in the flask, with stirring. (KMnO4 soln should be just sufficient to oxidize the ferrous salt, but it is well to add the last few ml in small portions.) Cool soln and dilute to 1 liter with H20.

b) Standard titanium trichloride soln. 

Add 100 ml of commercial 15-20% TiCl3 soln to 200 ml of HC1, boil 1 min., cool, and dilute to 4500 ml with H2O. Place soln in container with II atmosphere provision and allow to stand 2 days for absorption of residual 0. Preserve the TiCl3 soln in an atmosphere of H (Chap. 21, Fig. 27), taking care to have all joints air-tight, and covering stoppers (preferably countersunk) with suitable wax. Standardize by titrating 20 ml of the FeNH4(SO­4)2 soln against the TiCls soln in a protective stream of C02 , using 1 ml of 5% NH4CNS soln as indicator. 1 ml of 0.1 .V FeNH4(SO4)2 = 0.01545 g of TiCl3."
The determination of the ascaridole content by a distillation technique is not to be recommended for routine analyses, since the ascaridole is so unstable that the oil is apt to decompose with explosive violence should the temperature not be carefully controlled.
Dodge119 has suggested the use of a solution of sodium bisulfite to determine the ascaridole content of wormseed oils. The difficulty of determining the exact end point and the length of time required for a determination militates against the use of this technique.
In spite of its recognized deficiencies, the method of Cocking and Hymas, at the present time official in "The National Formulary," Eighth Ed., and "The British Pharmacopoeia," is probably the most useful for commercial analytical control laboratories.
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108 The Eucalypts," 2nd Ed. (1920), Sydney, Australia, p. 357.
109 Eleventh Revision, 251.
110 J. Am. Pharm. Assocn. 10 (1921), 836. m
111 Ibid. 28 (1939), 591,
112 Twelfth Revision, 319. (Now official in "The National Formulary," Eighth Ed., 136.)
113 Analysts* (1930), 180.
114 (1932), 303.
115 Pauly (sec Pharm. Arch. 7 (1936), 9) contends that the empirical factor, 0.00665, is actually too high, since he obtained a value of 110 per cent for a redistilled ascaridole fraction using this factor. Experiments carried out in the laboratories of Fritzsche Brothers, Inc., appear to confirm this fact.
116 6th Ed., 735.
117 AnalystSI (1926), 170.
118 J. Am. Pharm. Assocn. 28 (1939), 589.

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