N e w Spectrophotometric Determination of Unsaturated 3-Keto Steroids Using 4-Aminoantipyrine Hydrochloride Reagent E. P. SCHULZ, M. A. DIAZ, GULLIVER LOPEZ, 1. M. GUERRERO, HERIBERTO BARRERA, A. L. PEREDA, and AMADA AGUILERA Synfex, S. A., Mexico, D. F., Mexico b A newly developed spectrophotometric technique for the determination of 3-keto steroids ( 1 -ene, enolic 2formyl, 1,4-diene, 4-ene, or 4,6-diene) has been investigated. Trace levels of formulated steroids (0.01yo)were determined in macro size ( 1 gram) samples. The development of the chromophore with 4-aminoantipyrine hydrochloride reagent was achieved at ambient temperature in periods ranging from 20 to 285 minutes for the various steroids investigated. Minor changes in the degree of unsaturation in rings A and 6, as well as the nature of ring-B substituents, effect radical changes in the wavelengths of maximum absorbance. The technique i s simple, sensitive, accurate, and relatively free of interferences.
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When applied to formulation assays, the 4-I.SP reagent exhibited one apparent disadvantage; namely, the tendency to react with esterified hesitol anhydrides (Spans) and with the corresponding polyosyethylene derivatives (Tweens) of the above compounds. The developed method showed special merit in the analysis of steroid esters formulated as oil-injectables, since no separation of the steroid from excipients was necessary. Comparison with results obtained by a 2,4-dinitrophenylhydrazine gravimetric method ( I ) indicated the superiority of the 4-LIl.lPmethod. Using the 4-hhI' reagent, techniques have been developed for the analysis of fluocinolone acetonide formulated in a cream or ointment base; for testosterone esters or progesterone in oilinjectable formulations; and for oxymethalone formulated in multi-vitamin preparations. In conjunction with the blue-tetrazolium reagent (analysis of the steroid a-keto1 side chain) the 4-.1AP reagent (analysis of ring -4 and B st,ructural features) offers a new approach to both quantitative and qualitative analyses of steroids.
HE REPORT by Huttenrauch (2) that unsaturated-3-keto steroids react with the addition product of :t annous chloride and p-aminodiniethylaniline to form chromogens in the 415- to 455-mp range suggested the possible use of other amines as potential reagentz for the assay of unsaturated-3-keto steroids. Of the various amines examined, only 4-aminoantipyrine hydrochloride (4-
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EXPERIMENTAL
AIP) (4-aniino-l,5-dimethyl-2-pheny1-3pyrazdone hydrochloride) showed any potential for quantitabive steroid analysis. This reagent has proved to be an estremely sensitive and specific reagent for unsaturated-3-keto steroids. In one instance, as little as 4 pg. of oxymethalone was detectable, while one of the least sen4tive steroids tested (fluocinolone acetonide-21-monosodium dihydrogen phosphate) was detectable a t a 4 0 - p g . level. In addition to the sensitivity and specificity of the 4--4-IP reagent, the presented method has the desirable feature of chromophore development at ambient temperature. The reaction required periods ranging from 20 to 285 minutes. The observed niasima for the 22 steroids esamined ranged from 331 to 394 nip. Structural correlations for substituent contribution to the observed wavelengths are also presented. 1624
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ANALYTICAL CHEMISTRY
Apparatus. I-ltraviolet spectrophotometer with a hydrogen lamp and I-em. quartz cells. Reagents. 1.0 ml. of 12.V hydrochloric acid diluted to 100.0 mi. with anhydrous reagent grade methanol. 100 mg. of 4-aminoantipyrine hydrochloride diluted t o 25.0 ml. with the acidified methanol. Celite 503 (Johns-llansville) overlaid with 12.Y hydrochloric acid for a 24-hour ppriod, washed to neutrality with distilled water, Jvashed with ethyl acetate, and dried a t 100" C. after a final methanol wash. 10% aqueous (w.'v.) sodium chloride solution. .I solution of Tween-precipitant, prepared b y heating 1.0 gram of phosphotungstic acid (12W03.H3P04 .XH,O) and 5.0 grams of barium chloride with ca. 80 nil. of sodium chloride reagent for 15 minutes in a 100-ml. volumetric flask on a steam bath; adding 0.1 ml. of 12-l' hydrochloric acid to the cooled contents; diluting t o the mark with
sodium chloride reagent; and filtering the final solution through No. 42 filter paper. General Procedure
for
Creams.
Extract a n accurately weighed portion of cream (equivalent to ea. 1280 pg. of steroid) with 50.0 ml. of methylene chloride. Transfer both phases to a separatory funnel, and filter the lower phase through a sintered-glass funnel containing 5 grams of anhydrous sodium sulfate. Evaporate the solvent from a 25.0-ml. aliquot of the above filtrate. Add 25.0 ml. of methanol; heat' for 10 to 15 seconds on a steam bath to liquefy the lowmelting excipients; and stir for 15 minutes. Filter (without' vacuum) through another sintered-glass funnel containing 2 granis of anhydrous sodium sulfate. Treat 2 separate 5.0-ml. aliquots of the methanol ext'ract as follows: Stir 1 aliquot with 0.5 gram of the Celite reagent and 35.0 ml. of sodium chloride reagent for 5 minutes. Vacuum-filter through a sintered-glass funnel containing 0.5 gram ' of Celite reagent. Stir 30.0 ml. of the filtered aqueous extract with 30.0 ml. of methylene chloride for 5 minutes. Separate both phases, and stir the entire organic phase for 10 to 12 minutes with 30.0 ml. of the Tween reagent. Let stand for ea. 10 minutes. Transfer to a separatory funnel ca. 28 ml. of the methylene chloride phase, using a 10-ml. graduated pipet. Permit to stand for an additional 10 minutes in the stoppered funnel. Filter through a sintered-glass funnel cont'aining a mixture of 0.2 gram of the Celite reagent and 1 gram of anhydrous sodium sulfate. Evaporate to dryness exactly 15.0 ml. of the filtrate (residue .I). This residue contains an expected 48 pg. of the formulated steroid. Treat the second 5.0-ml. aliquot with 0.5 gram of the Celite reagent and 35.0 ml. of the Tween reagent. Proceed exactly as above to the point where 30.0 ml. of filtered aqueous extract has been stirred with 30.0 ml. of methylene chloride. Restir the separated organic phase for 10 to 12 minutes with 30.0 ml. of the sodium chloride reagent. Filter the methylene chloride phase through a sintered-glass funnel containing 1 gram of anhydrous sodium sulfate. Evaporate to dryness 15.0 ml. of filtrate (residue B ) . l h i s residue contains none of the formulated steroid, -4dd 3.0 nil. of the 4-aminoantipyrinc
hydrochloride reagent solution (4-AhP) to residues A and B , as well as to a residue containing 48 pg. of corresponding steroid reference standard. Determine the absorbance values a t the specified analytical maximum, using as a reference the prepared 4-AAP reagent. Correct the absorbance of residue A with that obtained for residue B, and relate the corrected absorbance to the intensity of the standard chromogen. General Procedure for Ointments. Reflux a n accurately weighed portion of formulation (equivalent to 512 pg. of steroid) for 5 minutes with 20.0 ml. of methanol. Treat t n o separate 5.0ml. aliquot5 of the filtered methanol extract exactly as described above for the cream procedure. The subsequent procedure for the development of the chromophore also remains the same. General Procedure for Oil-Injectables. Dilute a measured portion of the formulation (equivalent to 200 pg, of steroid) to 10.0 ml. with the 4-.iAP reagent. h d d 1 0 0 ml. of the reagent to a n equivalent amount of corresponding steroid reference standard residue. Determine both absorbance values, using as a reference the 4-.2AP reagent solution. Correct the absorbance of the formulated steroid chromophore for the reading obtained after diluting the same volume of formulation to 10.0 ml. with acidified methanol, using methanolic hydrochloric acid reagent as a reference. Determine all absorbance values at the analytical maximum of the particular steroid. Relate the corrected absorbance to the intensity of the standard chromophore. General Procedure for Tablets a n d Capsules. Extract a quantity of ground tablets or capsule content5 (equivalent to 2500 pg. of steroid) by stirring for 15 minutes with 100.0 ml. of chloroform. Add 5.0 ml. of 4--2AP reagent solution to the residue from a 2.0-ml. aliquot of the filtered extract. Add the same volume of reagent to an equivalent amount of corresponding steroid reference standard residue. Determine both absorbance values, using as a reference the prepared reagent. Correct the absorbance of the formulated steroid chromophore with the reading obtained after the addition of 5.0 ml. of HC1-acidified methanol reagent to the residue from a 2.0-ml. aliquot of the filtered extract, using HC1-acidified methanol reagent as a reference. Relate the corrected absorbance to that of the reference standard steroid. Determine all absorbance values a t the analytical maximum of the particular steroid. Procedural Techniques. I n a t tempting to apply t h e generalized procedures previously described, t h e analyst should adhere to certain common precautions and conditions. Because of the Pvtremely volatile nature of methylene chloride, one should exercise t h e normal precautions (eutractions in glass-stoppered s p t e m s by magnetic stirring, portionwise evaporation
