(11) Kolthoff, I. RI., St,ork, J. T., Analyst 80, 860 (1955).

(12) Laitinen, H. A., 'Chemical Anal- ysis," p. 115, hlcGraw-Hill, New 170rk, 1960.

(13) Laitinen, H. A,, Jerinings, W. P., Parks, T. I)., IND. E ~ G . CHEV., ANAL. En. 18, 355, 358 (1946).

(14) Lingane, J. J., Smdl, L. A., ANAL. CHEM. 21. 1119 (1949).

(15) Rleites; L., Ibid. , 20 , 084 (1948).

(16) Ramette, R. W., J . C h m . Educ.

(17) Samson, S., Anal. Chim. dcta 13, 37, 348 (1960).

473 (1955). (18) Sridell. A , . "Solubilities of Inorganic . ,

and Metal (jrganic Compounds," 3rd ed., 1'01. I, pp. 34-40, Van Nostrand, New York, 1940.

(19) Shain, I., Perone, S. P., 4 x a ~ . CHEM. 33, 325 (1961).

(20) Stock, J. T., Turner, W. R., Chpm. Ind. (London) 1961, 1710.

(21) Tsynov, G. A., Muraskina, I. I., Uzbeksk. Khim. Zh. 1960, 38; C.A. 56, 844% (1962).

RECEIVED for review March 7 , 1963. Accepted July 22, 1963. The authors express their thanks to the Fulbright Foundation for financial support of thls work in the form of a research scholarship granted to Zui-feng Lin.

Polarogralphic Monitoring of DifF erential Reaction Rates

Determination of A4-3-ketosteroids in the Presence of A1l4-3- ke toste roid s

ALAN F. KRIVIS and GEORGE R. SUPP Cenfral Analytical labo;atories, Olin Mathieson Chemical Corp., New Haven 4, Conn.

b Polarographic monitoring of dif- ferential reaction rate analyses has been studied. A determination of residual A4-3-ketostert3ids in A1s4-3- ketosteroids has been made by follow- ing a pseudo first-order semicarbazone reaction. A simple plct of log i for the semicarbazone as a function of time was used to obtain the current due to the reduction of the A4-3- <etosteroid spe- cies. Comparison of the current ob- tained to a calibraton curve per- mitted the estimation of the amount of residual A4-3-ketosterc~id present.

r HE AKALYSIS of mixtures containing r s i m i l a r types of compounds by

their differing rates of reaction with a reagent recently was found to be a widely applicable technique (14). Siggia and Hanna have been able to determine mixtures of alcohols (io), amines (6), hydroxyl content of po ypropylene g1y- cols ( 7 ) , and unsaturz ted compounds (11) . I n addition, g -aphical kinetic methods for analysis of mixtures were extended to separation methods such a? distillation (la), to decompoqition rates ( I S ) , and to 1)umas nitrogen analyses (1). Earlier work by Lee and Kolthoff (9) indicated that mix- tures of olefins and of carbonyl com- pounds could be analyzctd.

The highly active gliicocorticoid and anti-inflammatory ~l],~-3-ketosteroidi are synthesized from ~~-3-ketosteroids and, therefore, the res dual A4-3-keto- steroids must be dete*mined. Forist (4) described an excellent spectrophoto- metric method for residual A4-3-keto- steroids which involwd a two-hour rcaction with thiosemic:irbnzide.

It was felt that a more rapid deter- mination of residual 14-3-ketosteroids in AS4-3-ketosteroids might be devised by using the graphic kinetic approach, and a study of the problem was under- taken.

EXPERIMENTAL

Reagents. All inorganic chemicals were reagent grade and were used without further purification.

T h e steroids used in this s tudy were obtained from K & K Laboratories, Inc. ,411 other organic chemicals were Eas tman White Label grade.

A combination buffer and reagent mixture containing, at final dilution, 0.1M semicarbazide hydrochloride, 0.0.5X acetic acid, 0.05M sodium ace- tate, and 0.0057, gelatin in 50% methanol was used.

Apparatus. A h1etrohm Polarec- ord E-261 R was used in conjunction with a thermostated (25" + 0.1' C.) H-cell containing a saturated calomel reference electrode (8) to obtain the polarographic data. The damping was set at position 5, and the current and, when necessary, potential values were determined graphically, using the av- erage of the recorder traces.

The capillary used had a constant of 2.049 mg.2'3t1'6 at 25 cm. of Hg in dis- tilled water at open circuit.

Procedure. All solutions were equi- librated at 25' C. and deoxygenated with purified and equilibrated nitrogen prior t o mixing.

T h e required amount of buffer solu- tion and methanol was transferred t o the polarographic cell and deoxygen- a ted for 10 minutes. T h e requisite volume of deoxygenated steroid stock solution was then injected into the solution in the cell and mixed thor- oughly, and the current at the potential for the semicsrhsxone reduction (- 1.12

volts) was measured as a function of time. A separate calibration curve for the desired semicarbazone was pre- pared in order to relate current t o concentration for the unknown runs. The calibration data mere obtained by adding known amounts of the A4-3- ketosteroid to the buffer solution, per- mitting the reaction t o go to completion, and then measuring the current for the semicarbazone reduction. h plot of current vs. steroid concentration was made from the data.

The steroid analyses mere performed by plotting log i us. time and extrap- olating the line for the slower reacting species t o time zero; the extrapolated current was the total current due t o the reduction of the faster reacting material. The current value was then related t o coricentration of the Pteroid via the calibration curve described above.

RESULTS AND DISCUSSION

Mixtures of carbonyl compounds were analyzed by utilizing a second-order oximation reaction (10) with the rate data obtained by potentiometric ti- trations. The titration method, how- ever, was time-consuming and cumber- some, and a polarographic approach with continuous monitoring appeared to offer advantages.

An oximation reaction mas carried out with a mixture of A I 4 - and A4-3- ketosteroids. It was found that the reaction was too slow for the purposes of the analysis and attempts to speed it up by increasing the hydroxylamine concentration and adding a catalyst (5) mere not successful. However, mix- tures of other carbonyl compounds could be analyzed using the second-order oximation reaction.

VOL. 35, NO. 10, SEPTEMBER 1963- 0- 141 1

Table 1. Polarographic Differential Reaction Rate Analyses of Mixtures of

Hydrocortisone and Prednisolone

Hydrocortisone Added,a mg. Found, mg.

0.017 0.01s 0.017 0.022 0.020 0,019 0.021 0,015 0.022 0,022 0.02s 0.028 0.039 0.039

0.024 0.020 0.025 0,020 0.039 0.030

0. u39 0.034 0.044 0 , 0 3 1 O.O!)P 0. 0SY

In the presence of 1.78 mg. of prednis-

b In the presence of 1.73 mg. of prednis- olone.

olone.

10

3

8

I

6

5

4

%

u 3

>

I

Table II. Polarographic Differential Reaction Rate Analyses of Mixtures of

Cortisone and Prednisone

Cortisone Added,” mg. Found, mg.

0.014 0.014 0.019 0.023 0.024 0.024 0.026 0.029 0.029 0,030 0.034 0.048

0.015 0.021 0.020 0.030 0.02% 0.032 0.027 0.022 0.022 0.026 0.027 0.031

5 In the presence of 1.77 mg. of pred- nisone.

Semicarbazone formation, although sensitive t o the reaction medium, has been reported to be very rapid with half lives for many carbonyl compounds in the neighborhood of one minute or less (2). The rapidity of semicarbazone formation permitted the determination of individual carbonyl compounds in the atmosphere (S), using a polaro- graphic end point. These reports in- dicated that steroid semicarbazones might be produced rapidly enough to permit a differential analysis. Further- more, the use of a large excess of semi- carbazide reagent to give pseudo first- order kinetics could simplify the cal- culations needed for the analysis.

The integrated form of the equation for a first-order reaction is

In (Co/C) = kt (1)

Figure 1 . Polarographic differential rate analysis of hydrocortisone in prednisolone

or

In C = In Co - kt (2)

where Co is the concentration of the re- actant a t time zero and C is the con- centration a t time t . A plot of In C (or log C) us. t produces a straight line with a slope of -k (or -k/2.303).

I n the present instance, the concen- tration of the carbonyl compounds can be measured as a function of time but it was far more convenient to measure the current for the semicarbazone formed. Since the current is directly proportional to concentration, a plot of log i us. t gave a straight line. With a mixture of two compounds reacting at different rates, two distinct slopes were obtained. Extrapolation of the second slope to zero time gave a current value which was due to reduction of only the faster reacting species.

Mixtures of 4-pregnene-I 1 p, 17a,21- triol-3,20-dione (hydrocortisone) and 1,4 - pregnadiene - 11&17a,21 - triol- 3,20-dione (prednisolone) and of 4 - pregnene - 17a,21 - diol - 3,11,20- trione (cortisone) and 1,4-pregnadiene- 17a,21-diol-3,11,20-trione (prednisone) were reacted with an excess of semi- carbazide hydrochloride and the current for the semicarbazones plotted on a semilogarithmic chart as a function of time. Figure 1 shows the curve for a mixture of 5% hydrocortisone and 95% prednisolone and indicates the simplicity

of the procedure. Table I lists the results obtained for various hydro- cortisone-prednisolone mixtures and Table 11, the analyses for cortisone- prednisone mixtures. The mean of the errors for the hydrocortisone-pred- nisolone mixtures was 15.5% with a standard deviation of 111.0. The mean of the errors for the cortisone- prednisone mixtures was 18.8% with a standard deviation of k14.6. The artificial mixtures ranged in concentra- tion from less than 1% to 5% of the A4-3-ketosteroid component with a total sample size of about 2 mg. At the lower levels studied, the faster reac- tion was essentially complete within threc to five minutes and sufficient data could be obtained within ten minutes to perform the analysis.

values for the steroid semicar- bazones varied from -1.10 volts to -1.16 volts for the four compounds studied. In order to simplify the pro- cedure, all current measurements were made at an applied potential of -1.12 volts. The potential used was chosen also to avoid any contribution to the current from the reduction of the steroid ketones themselves.

ACKNOWLEDGMENT

The authors thank Sidney Siggia and 3. G. Hanna for many helpful discussions and suggestions.

LITERATURE ClTED

(1) Block, J. Morgan, E., Siggia, S.,

(2) Conant, J. P., Bartlett, P. D., J . Am.

(3) Coulson, D. M., Anal. Chim. Acta 19,

(4) Forist, A. A., AKAL. CHEM. 31, 913

( 5 ) Fritz, J. S., Yamamura, S. S., Brad-

(6) Hanna, J. G., Siggia, S., Ibid. , 34, 547

ANAL. CHEM. 35, 573 (1963).

Chem. SOC. 54, 2881 (1932).

284 (1958).

(1959).

ford, E. C., Ibid.,.p. 260.

( 1 962). \ - - - , (7) Hanna, J. G., Siggia, S., J . Polymer

(8) Komyathy, J. C., Malloy, F., Elving,

(9) Lee, T. S., Kolthoff, I. M., Ann. N . Y .

Sci. 56, 297 (1962).

P. J., ANAL. CHEW 24,431 (1952). ~I

Acad.’Sci. 53, 1093 (i951).’

CHEM. 33, 896 (1961).

N. M., Ibid., p. 362 (1963).

(10) Siggia, S., Hanna, J. G., ANAL.

(11) Siggia, S., Hanna, J. G., Serencha,

(12) Ibid., p. 365. (13) Ibid., p. 575. (14) “Symposium on Use of Differential

Reaction Rates to Analyze Organic Mixtures,” Abstracts, Division of Ana- lytical Chemistry, 142nd Meeting, ACS, Atlantic City, N. J., 1962.

RECEIVED for review February 5, 1963. Accepted June 14, 1963.

141 2 ANALYTICAL CHEMISTRY