Feb., 19501 The METHACRYLATE POLYMERS AND CO-POLYMERS Determination of Phenodoxone in 71 Urine BY J. E. PAGE AND HEATHER KING SYNOPSIS-A colorimetric method has been devised for determining pheno- doxone in urine. Phenodoxone is the official name of a recently available powerful analgesic, DL-6-morpholino-4 : 4-diphenylheptan-3-one, the hydro- chloride of which is available under the proprietary name “Heptalgin.” The base from urine samples containing about 0.4mg. of the drug is liberated with alkali and extracted into toluene; the toluene extract is shaken with an aqueous solution of bromophenol blue buffered a t pH 4.0. An amount of dye, equivalent to the weight of drug in the toluene layer, is carried over into the toluene as a yellow compound, and this is decomposed by shaking the toluene layer with aqueous alkali.The sodium salt of the dye then enters the aqueous layer and is determined colorimetrically. Basic substances that react with bromophenol blue to form a toluene- soluble compound (alkaloids, such as codeine, and synthetic analgesics, such as pethidine and amidone) interfere with the determination. PHENODOXONE is the official name given to DL-6-morpholino-4 : 4-diphenyheptan-3-one. The hydrochloride of this substance was first studied under the description CB11, which will be used for convenience in the present communication; it is now generally available under the proprietary name “Heptalgin.” I t is used because of its marked action as an analgesic, which is combined with a relatively low toxicity, so that its therapeutic ratio is higher than that of other similar compounds.Its preparation has been described by Dupr6, Elks, Hems,72 PAGE AND KISG: THE DETERMINATION OF [Vol. 75 Speyer and Evans1 and by Attenburrow, Elks, Hems and Speyer,2 and its pharmacology by Basil, Edge and Somer~.~ For the determination of CBll in urine we have used a modification of Lehman and Aitken’s procedure4 for determining pethidine. Scott and Chen6 and Cronheim and Ware6 have employed like methods for the estimation of amidone and Hopewell and Page’ have described a somewhat similar technique for the determination of long-chain aliphatic amines. Ethyl- 1 -methyl-4-phenylpiperidine-4-carboxylate ,CH,.CH 2,c/C0. OCH ,. C H, \CH,.CH,/ \c> hydrochloride. Pethidine. CH,.N HC1 6-Dimethylamino-4 : 4-diphenylheptan-3-one hydrochloride.Amidone. CH/ 6 Morpholino-4 : 4-diphenylheptan-3-one hydrochloride, “ Heptalgin ” (CBll). n Fig. 1 The method depends on the reaction of equimolecular quantities of the analgesic and bromophenol blue to form a toluene-soluble compound. The free base of the drug is extracted into toluene and the toluene extract is shaken with an aqueous solution of the dye buffered at pH 4.0. An amount of dye, equivalent to the weight of drug in the toluene layer, is carried over into the toluene as a yellow compound, which is decomposed by shaking the toluene with aqueous alkali, whereupon the sodium salt of the dye enters the aqueous layer and is determined colorimetrically. Under these conditions, CBll gives a more intense colour than when tested by either Lehman and Aitken’s bromothymol blue method4 or Cronheim and Ware’s bromocresol purple method.6 The polarographic behaviour of CBll was also studied with a view to finding a suitable analytical method.In 0.1 N potassium chloride it gave a polarographic step with a charac- teristic peak at -1-75 v. (cf. Fig. 2). The step height was approximately proportional to concentration over the range 0.0075 to 0.075 per cent. However, the step height was sensitive to small amounts of surface-active material and appeared at a relatively high potential, so that the method was unsuitable for determining CBll in biological fluids. Experimental firocedure-Our recommended procedure for the determination of CB 11 is as follows. Shake 10 ml. of the solution, containing about 4 mg.of CBll per 100 ml., with 2 ml. of 10 N sodium hydroxide and 30 ml. of redistilled toluene in a separating funnel. Pipette 25 ml. of the supernatant toluene into a second separating funnel and shake for 5 minutes with 20 ml. of a bromophenol blue solution buffered at pH 4-0 (made by mixing 60 ml. of 0.05 M potassium hydrogen phthalate with 40ml. of a 0.08 per cent. bromophenol blue solution). Centrifuge (if necessary) the mixture from the separating funnel, transfer 20 ml. of the supernatant toluene to a third separating funnel, and extract in turn with three 5-ml. portions of 0.1 N sodium hydroxide. Dilute the combined alkaline extracts to 20ml. with 0.1 N sodium hydroxide and examine an aliquot portion in a Hilger photo-electric absorptio- meter fitted with a yellow gelatin filter (Ilford No.606) and a heat-resisting Chance glassFeb., 19501 PHENODOXONE IN URINE 73 filter (No. H503). The concentration of CBll can be determined from a calibration curve prepared for solutions containing between 1.0 and 10.0 mg. of CBll per 100 ml., over which range there is a linear relationship between drug concentration and absorptiometer reading. Solutions containing more than 10mg. of analgesic per 100ml. should be diluted before testing. Determinati0.n in zcrine-The bromophenol blue method may be used to determine CBll in human and rat urine, but because any basic substance capable of reacting with bromophenol blue to form a toluene-soluble compound will respond to the test, the following precautions must be observed.I lo: I = ‘0 I: I’ I 1.4 1.6 I -8 2.0 POTENTIAL IN’ VOLTS. Fig. 2. Polarogram for an 0025 yo solution of CBll in 0.1 N (Drop-time on open circuit in 9.1 N Weight of potassium chloride solution. potassium chloride solution at 26’ c.=3.13 SBCS. mercury dropping per sec. = 1.82 mg.) Certain alkaloids such as codeine, and sFthetic analgesics such as pethidine and amidone, interfere with the determination; the intensity of the colour formed by amidone is greater than that produced by an equivalent quantity of CB11, and is about the same as that formed when amidone is examined by Cronheim and Wue’s procedure.6 Adequate control specimens must therefore be tested and particular care must be exercised in analysing urine samples from subjects who have received special medication.Bacterial growth in the urine leads to the production of basic substances giving the same colour response as CB11; a small amount of an antiseptic (e.g., mercuric chloride or toluene) must therefore be placed in the vessel used for collecting the urine. During the solvent extraction of urine, the ratio of the volume of toluene to that of the urine must be kept as high as possible in order to prevent the formation of stable emulsions. The calibration curve for the determination of CBll in urine is about 30 per cent. lower than that for its determination in simple aqueous solution. REFERENCES 1. Dupre, D. J., Elks, J., Hems, B. A., Speyer, K. N., and Evans, R. M., J. Chem. SOC., 1949, 500. 2. Attenburrow, J., Elks, J., Hems, B. A., and Speyer, K. N., Ibid., 1949, 610. 3. Basil, B., Edge, N. D., and Somers, G. F., Brit. J. Pharmacol., in the press. 4. Lehman, R. A., and Aitken, T., J. Lab. Clin. Med., 1943, 28, 787. 6. Scott, C. C., and Chen, K. K.. J. Pharmacol., 1946, 87, 63. 6. Cronheim. G., and Ware, P. A., Ibid., 1948, 92, 98. 7. Hopewell, B. M. C., and Page, J. E., Analyst, 1946, 70, 17. RESEARCH DIVISION GLAXO LABORATORIES LTD. GREENFORD , MIDDLESEX May, 1949