These factors enable the modification of the method to differentiate the phosphine present in putrefied tissue samples, metal phosphide, and white phosphorus by performing the experiment under these different conditions. While zinc phosphide has been used as standard for phosphorus in this work, white phosphorus can also be used if the analysis for white phosphorus is required. The experimental conditions can then be adjusted to detect only white phosphorus. Standards such as ammonium phosphate, normally used in neutron activation analysis for phosphorus, are not recommended for use in this work since they cannot produce phosphine in the distillation step and, therefore, cannot be treated in the same way as the sample. Further, it was found in preliminary experiments, that if they were used as a standard, low values were obtained for the amount of phosphors added to the tissue. This could, however, be due to incomplete production, distillation, and collection of phosphine from the zinc phosphide added to the tissue. In using the neutron activation analysis technique, the phosphorus can be easily and uniquely identified. The chemical separation procedure using phosphorus carrier ensures the separation of 32P. The half-life of 32Pcan be determined. The energy of the /3 rays can be measured by using aluminium absorbers and constructing a Feather plot. This is an important improvement over Curry’s method where the recognition of a positive reaction for phosphorus may be difficult when only nanogram quantities of the phosphorus are present.
Sensitivity. Under the irradiation conditions used in this
work, the neutron activation technique can detect phosphorus down to 5 ng. However, the actual limit of detection of phosphorus in tissue by using the entire procedure is set by the background 32P radioactivity obtained due to contamination from the materials used. Results from 15 experiments performed with tissue samples containing no toxic phosphorus indicated that, by using redistilled analar reagents and recrystallized analar chemicals, this blank phosphorus radioactivity can be reduced to the equivalent of 7 ng. Thus, taking this into consideration, the practical lower limit of detection of toxic phosphorus by this method can be set as approximately 10 ng. This is an improvement of a factor of 10 compared to the best presently available technique (2). Therefore, when only small quantities of samples are available or when only small amounts of phosphorus are expected to be present in the sample, this method is very useful, effective, and quantitative. This advantage and the capability of the method to identify uniquely phosphorus-32 is important in medico-legal applications. ACKNOWLEDGMENT
The authors express their sincere appreciation to the late H. W. Smith of this laboratory for his continued encouragement and criticism.
RECEIVED for review May 6, 1968. Resubmitted January 27, 1970. Accepted January 27,1970.
Diethyl Oxalate as New Reagent for Spectrophotometric Determination of Ketosteroids Sandor Gorog Chemical Works G . Richter, Budapest X . , Hungary
A new spectrophotometric method has been developed for determination of A4-3-keto-, 17-keto-, and 20-ketosteroids which is based on the Claisen condensation of the active :methylene groups of ketosteroids with diethyl oxalate leading to spectrophotometrically active glyoxalyl derivatives. The development of the chromophore was carried out at 0 O C or at room temperature in a mixture of tertiary butanol and cyclohexane in the presence of sodium tertiary butoxide while the absorbance was measured in moderately acidic ethanol. The method is suitable for the characteristization and quantitative determination of ketosteroids, particularly in the assay of mixtures and pharmaceutical ketosteroid formulations. The relative standard deviation of the method is =tl.O-l.9% for pure ketosteroids and 152.1% for formulated ones.
A LARGE NUMBER of spectrophotometric methods are in use for the determination of ketosteroids, most of them measuring the light absorption of the Schiff base or hydrazone formed using strongly acidic solutions of aromatic amines [e.g., 4-amino antipyrine ( I ) ] or hydrazides [e.g., isonicotinic acid hydrazide (211 as reagents. A considerable proportion of (1) E. P. Schulz and coworkers, ANAL.CHEM., 36, 1624 (1964). (2) E. J. Umberger, ibid., 27, 768 (1955). 560
ANALYTICAL CHEMISTRY, VOL. 42, NO. 6, MAY 1970
these methods is suitable only for the determination of unsaturated ketosteroids. Procedures which were carried out in an alkaline medium have been preferred in many cases (e.g., determination of ketosteroids in the presence of their acid-sensitive derivatives such as ketals, vinyl ethers etc.). The determinations carried out in alkaline media are based on the reactions of the active methylene groups in the vicinity of the keto group. The Merent variations of the Zimmermann method (3) are used most frequently where m-dinitrobenzene or other aromatic di- or trinitro (4) derivatives are used as the reagents. Among the other methods, the procedure based on the reaction with 2,6-di-tert-butyl-p-cresoldescribed by Schulz (5) is worth mentioning. The Zimmermann method is used mainly in biological analysis; hence, there are only a few examples of its use in pharmaceutical analysis (6). The 2,6-di-tertbutyl-p-cresol method although used in pharmaceutical (3) W. Zimmermann, “Chemische Bestimmungsmethoden von
Steroid-hormonen in Korperfliissigkeiten,” Springer, BerlinHeidelberg-Gottingen, 1955, pp 53-57, 63-76. (4) M. Pesez and J. Bartos, Ann. Pharm. France, 22, 541 (1964). ( 5 ) E. P. Schulz and J. D. Neuss, ANAL.CHEM., 29, 1662 (1957). (6) J. F. Chissel, J. Pharm. Pharmacol., 16, 490 (1964).
analysis (7), is empiric in character, its mechanism is a subject for debate (8, 9 ) and its reproducibility has also been criticized (10). This paper presents a newly developed method which utilizes a diethyl oxalate reagent in alkaline solution. Under the given conditions, Claisen condensation takes place transforming the ketosteroids quantitatively to glyoxalyl derivatives which have characteristic and intense light absorption, suitable for the characterization and quantitative determination of both saturated and unsaturated ketosteroids. EXPERIMENTAL Apparatus. A “Spectromom 202” ultraviolet spectrophotometer with 1-cm quartz cells was used in this study. Reagents. SOLVENTMIXTURE.Mix 900 ml of reagent grade tertiary butanol, distilled over sodium, with 100 ml of reagent grade cyclohexane. The water content of this mixture should not exceed 0.05% as examined by the Karl Fischer method. SODIUM BUTOXIDE REAGENT, 0.25N. Dissolve 2.88 grams of sodium in 400 ml of the above mixture of tertiary butanol and cyclohexane by boiling and dilute the solution to 500 ml. If sodium butoxide begins to crystallize, as it sometimes does, warm the reagent gently before use. The reagent can be used for several months if stored in a well-closed bottle. Use the reagent until its sodium hydroxide content does not exceed 3 0 z of the total basicity. Determine the sodium hydroxide content in the following manner. Add Karl Fischer reagent to a mixture of 3 ml of dehydrated pyridine and 0.2 ml of acetic acid until a brown color appears. Add 5 ml of the reagent to be tested and titrate the solution with the Karl Fischer reagent to the above brown color. Calculate the normality of sodium hydroxide from the following equation : N
=
0.0111 X K.F. reagent consumption X water equivalent
DIETHYL OXALATE REAGENT,1M. Dilute 73 grams (67.3 ml) of redistilled diethyl oxalate to 500 ml with the above described mixture of tertiary butanol and cyclohexane. METHANOL, reagent grade was used. ETHANOL, reagent grade was also used. HYDROCHLORIC ACIDwas 0.5N aqueous solution. General Procedure. Dissolve an accurately weighed quantity of the sample equivalent to about 0.01 g of ketosteroid in the solvent mixture and dilute this solution in a volumetric flask to a volume of 25 ml. Transfer 2.0 ml of this stock solution to a carefully dried 50-ml volumetric flask. Add 2 ml of cyclohexane, 0.5 ml of diethyl oxalate reagent, and finally 2.5 ml of sodium butoxide reagent. Allow the mixture to stand in the stoppered flask at room temperature for 15 minutes, then add 3 ml of 0.5N hydrochloric acid and dilute to volume with ethanol. Determine the absorbance of this solution against a similarly treated blank at the analytical maximum of the reaction product of the particular ketosteroid (see Table I). Calculate the ketosteroid content on the basis of the absorbance of a similarly treated standard solution. Procedure for Oil-Injectables. The formulations investigated were estrone acetate, 5 mg/ml; 19-nortestosterone decanoate, 25 mg/ml; and progesterone, 12.5 mg/ml containing 2.5 mg/ml estradiol benzoate. Solvent employed was oleum helianthi. ~~
(7) S. Ansari and R. A. Khan, J. Pharm. Pharmacal., 12, 122 (1960). (8) J. Bartos, Ann. Pharm. France, 17,141 (1959). (9) E. P. Schulz, M. A. Diaz, and L. M. Guerrero, J. Pharm. Sci., 53, 1119 (1964).
(10) D. C. Garratt, “The Quantitative Analysis of Drugs,” 3rd ed., Chapman and Hall, London, 1964, p 599.
Table I. Spectral Data of Some Ketosteroids after Treatment with Diethyl Oxalate Molar Maximum, absorptivity Ketosteroid mCc A4-3-Ketosteroids 7580 245 Estr-4-ene-17P-ol-3-0ne 4900 317 (19-nortestosterone) 244 7340 19-Nortestosterone 4950 317 phenylpropionate 244 7400 19-Nortestosteronedecanoate 316 4940 7850 244 19-Nor-17a-pregn-4-ene-20-in318 4960 17P-ol-3-one(norethisterone) 7360 246 Norethisterone acetate 318 4990 252 7340 Androst-4-ene-17P-ol-3-0ne phenyl6660 propionate (testosterone phenyl324 propionate 251 7310 Testosterone isocaproate 6730 324 75 IO 17a-Methyl-androst-4-ene- 17P-01252 3-one (methyltestosterone) 6720 324 17-Ketosteroids Androst-5-ene-3P-01-17-one 10710 294 (dehydroepiandrosterone)
Dehydroepiandrosterone acetate Androst-1,4-diene-3,17-dione
294 294 289
10400 15500 10950 11680
289 289
11850 11620
290 292
9870 10130
244
Estr-1,3,5(lO)-triene-3-01- 17-one (estrone) Estrone methyl ether Estrone acetate= 20-Ketosteroids Pregn-5-ene-3P-ol-20-one 16a,17a-Epoxy-pregn-5-ene-3P-01-
20-one acetate Pregn-5,16-diene-3P-ol-20-one 309 acetate A4-3,17-Diketosteroids Androst-Cene-3,17-dione 297 Estr-4-ene-3,17-dione 296 A4-3,20-Diketosteroid Pregn-4-ene-3,aO-dione 293 (progesterone) a It hydrolyzes during the condensation reaction.
11850 14900 14300 14010
Vigorously shake 1.0 ml of the formulation for 5 minutes with 50 ml of methanol in a well-stoppered flask. Allow the mixture to stand until the oil has settled. Evaporate an aliquot of this extract to dryness-10 ml in the case of estrone acetate, 5 ml in the case of 19-nor-testosterone decanoate and 2 ml in the case of progesterone. The subsequent procedure for the development of the chromophore is the same as that described above, the only difference being that the reaction is carried out under ice-cooling conditions for 45 minutes. Evaporate to dryness another aliquot of the extract of the same volume as in the case of the test solution, dissolve the evaporation residue in ethanol, and add this solution to the blank after it has been acidified with hydrochloric acid. Procedure for Determination of Methyltestosterone in Tablets. Extract about 0.8 g of finely powdered tablets, containing 4 mg of methyltestosterone per 0.3 g tablet, by boiling with 15 ml of cyclohexane. Filter, wash the filter with cyclohexane, and dilute the filtrate to 25 ml. Transfer 2.0 ml of the extract to a 50-ml calibrated flask, and 2 ml of the solvent mixture, and proceed exactly as described under General Procedure. Procedure for Determination of Androst-5-ene-3P-ol-17-one Contamination in 17a-Methyl-androst-5-ene-3P,17P-diol. Dissolve about 0.05 g of the sample in a mixture of 2 ml of the solvent mixture, 2 ml of cyclohexane, and 2.5 ml of sodium butoxide reagent and proceed as described under General Procedure. ANALYTICAL CHEMISTRY, VOL. 42,
NO. 6, MAY 1970
561
f2000
c 8000
4000
220
260
340
300
A
380
420
m !
Figure 1. Absorption spectra of some ketosteroids after treatment with diethyl oxalate
.-*-.-.-.-.-.-.-. _ . -
Methyltestosterone Dehydroepiandrosterone _____--__--Estrone - . - . - . - . - Pregn-S-ene-3-01-20-0ne -X -X -X -X - Progesterone RESULTS AND DISCUSSION Determination of Optimum Conditions for the Reaction. The condensation of steroid ketones with diethyl oxalate is often used in steroid syntheses. According to the literature, the corresponding glyoxalyl derivatives which have characteristic and intensive light absorption in the ultraviolet region are formed in almost quantitative yield. This encouraged us to investigate the possibility of the analytical use of this reaction. A large number of glyoxalyl derivatives have been prepared and their spectral data are described in the literature-e.g., (11, 12) and a great number of patents. Thus we were able to predict the position and intensity of the analytical absorption band, determine the conditions suitable for the stochiometric and quantitative operation of the condensation reaction. Solvent and Reagent Concentration. Tertiary butanol has been found the best solvent for the condensation reaction. To prevent it from freezing at room temperature, we mixed it with 10% of cyclohexane. As in certain cases it is necessary to carry out the reaction at 0 "C, further cyclohexane has been added to avoid the complete freezing of the reaction mixture. Diethyl oxalate and the condensing agent, sodium butoxide, are used in a very large (more than 100-fold) excess. This enables the use of solvents which are not completely anhydrous-Le., with a water content not in excess of 0.05%. Solvents with a greater water content cannot be used as water transforms a stochiometric amount of sodium butoxide to sodium hydroxide, and the latter causes the rapid hydrolysis of diethyl oxalate. Meeting this requirement is the most ~~
(11) H. M. Kissman, A. M. Small, and M. J. Weiss, J. Amer. Chem. SOC.,82, 2312 (1960). (12) G . R. Allen and M. J. Weiss, ibid., p 2840. 562
ANALYTICAL CHEMISTRY, VOL. 42, NO. 6, MAY 1970
important condition of the reproducibility of the recommended method. Effect of pH. Glyoxalyl derivatives are present as enolates in alkaline media and in undissociated enolic form in moderately acidic media. The latter conditions proved to be most suitable for analytical purposes, thus the absorbance measurements have been carried out at about pH 2. The spectra of the glyoxalyl derivatives investigated are independent of the pH in the pH range 1.5 to 2.5. Effect of Reaction Time and Temperature. Room temperature, about 25 "C,and a reaction interval of 15 minutes were chosen for the development of the chromophore. The reaction is almost quantitative within 5 minutes and the changes in the spectra remain within experimental error if the solution is allowed to stand for an additional 1 hour after 15 minutes reaction time. In certain cases-e.g., investigation of oil-injectables-it was necessary to carry out the reaction at 0 "C. A reaction time of 45 minutes is needed for complete reaction at this temperature. Chromophore Stability. After acidification, the spectra remain constant for at least 24 hours. Chromophore Linearity. Beer's law has been found valid in each instance within the 0.01-1.0 absorbance range. Blank Solution. As diethyl oxalate is inclined to react with the possible impurities of the solvents under the given conditions and as self-condensation may also occur, the blank must contain the same reagents as the test solution and must be treated similarly. The absorbance of the reagent blank is relatively low, after the 15 minutes reaction period 0.128 at 290 mp, 0.048 at 310 mp, and 0.029 at 330 mp. Its dependence, however, on the reaction time and quality of reagents cannot be neglected.
Investigation of Oil-Injectables. Direct methanolic extraction which worked satisfactorily in our earlier studies (13) has been applied to the investigation of oil-injectables. In these cases an appropriate aliquot of the methanolic extract was added to the blank, but this was after the acidification step in order to avoid interaction between diethyl oxalate and the ketosteroid. The error caused by the absorbance of methanol-soluble components of the oil can thus be cancelled out. The interaction of these components with diethyl oxalate could be avoided by carrying out the condensation at 0 "C. Steroids Investigated. Table I summarizes the spectral data of the steroids investigated after reacting with diethyl oxalate. The spectra of some characteristic ketosteroids after the above treatment are shown in Figure 1. Most of the compounds investigated belong to the pharmaceutically or industrially important Ad-3-keto-, 17-keto-, and 20-ketosteroids such as 17a-methyl-androst-4-ene-17P-01-3one (methyltestosterone), androst-5-ene-3P-ol-17-one(dehydroepiandrosterone), pregn-5-ene-3@01-20-one,etc. The reaction equation and the formulas of the condensation products for these compounds are seen in Scheme I.
Table 11. Ketosteroid Assay of Various Formulations Ketosteroid content Formulation Nominal Found" Estrone acetate oil-injectable 5 mg/ml 5.11 mg/ml Progesterone oil-injectable 12.5 mg/ml 12.41 mg/ml 19-Nortestosterone decanoate oil-injectable 25 mg/ml 25.31 mg/ml Methyltestosterone tablet 4 mg/tablet 3.94 mg/tablet Mean value of six determinations.
Re1 std dev, 7% ~t2.1 d11.9 1.9
=t
f1.5
Table 111. Determination of Dehydroepiandroesterone, DEA, in 17a-Methyl-androst-5-ene-3p,17~-diol, MAD DEA content in MAD Taken, Found, 0.13 0.11 0.27 0.27 0.40 0.43 1.29 1.25 2.50 2.39
z
z
OH
Methyltestosterone
0 I
& OH
CcO-C,H,
t 0
O
OH
W
2-Glyoxalyl methyltestosterone
OH 0
I
II
HC = c - c - 0
-c,y
I c=o
HO
-
!6 Blyoxalyl dehydro epi
-
2f
- G/yoxaly/ pregnenolone
androsterone
Scheme I Scope and Limitation. The data in Table I show that the method is suitable for the characterization and quantitative determination of 17-ketosteroids, A4-3-ketosteroids, and 20-ketosteroids which are unsubstituted at C-16, (2-2, and C-21, respectively. In the case of A4-3-ketosteroids, from among the two absorption maxima the long One has been used for quantitative purposes. (13) S. Gorog,J. Pharm. Sci., 57, 1737 (1968).
Saturated 2-keto steroids form 2,4-bis-glyoxalyl derivatives; this reaction, however, needs a longer reaction time. A1~4-3-ketosteroidsdo not react with diethyl oxalate, which enables the selective determination of Ad-3-ketones in the presence of A1,4-3-ketocompounds. (The details of this problem will be treated elsewhere.) A 4-3-ketosteroids with a corticosteroid side chain (such as hydrocortisone, etc.) give a positive reaction. However, the reproducibility of the results was not good enough to include them in Table I. The reason for this is the sensitivity of the a-keto1 side chain in alkaline media. A 4-3,17-diones(e.g., androst-4-ene-3,17-dione) and A 4-3,20diones (e.g., progesterone) form bis-glyoxalyl derivatives. Keto groups in other positions are of less importance and seldom occur alone; therefore they are not examined here. In addition to the applications described, the recommended method has been successfully used for a number of other problems (e.g., determination of ketosteroids in fermentation liquor, control of the reduction of ketosteroids and oxidation of steroid alcohols). Some special applications and the generalization of the method for the examination of nonsteroid oxo-compounds will be treated elsewhere. Precision of the Method. The molar absorptivities listed in Table I are the mean values of 3-4 measurements. In the case of some characteristic ketosteroids, six parallel measurements were carried out. The statistical analysis of the latter results showed a standard deviation of +1.5% for 19-nortestosterone, + 1.9% for norethisterone, f1.0% for testosterone phenylpropionate, i1.3% for dehydroepiandrosterone acetate, i1.1% for pregnenolone, and 1.6% for progesterone. The results of the analysis of several formulated keto steroid pharmaceuticals are summarized in Table 11. Table I11 shows the results of analysis of 17a-methylandrost-5-ene-3& 17P-diol samples with added dehydroepiandrosterone.
*
ACKNOWLEDGMENT
The author thanks Miss M. Kapds for technical assistance.
REcEIVED for review September 23, 1969. Accepted January 28, 1970. This paper is the 14th in a series on Analysis of Steroids. ANALYTICAL CHEMISTRY, VOL. 42, NO. 6, MAY 1970
563
