crivctte and indicatcs that 100 pg. of the steroid produrrs an absorbance of 0.76 unit in 54 minutes. From this information it would seem possiblo to identify and quantitate a very small amount of the strroid. The prochict acrumula t r s iiniformly with time and stwoid ronrrntration, so i t can be used to qunntitatr the concentration of strroids in iinknown samples. There is no shifting in the 380-mp peak, whirh indicates t ha t the reaction can be ca- pably analyxed during this period.

Subsequent to the study of known ketosteroids, various test samples were stridicd in an attempt to extract, purify, identify, and quantitate the compounds isolated by using the 7' >immermann reaction in conjunction with repetitive scanning. A placental extract was pre- pared and progesterone was purified by the method desrribed by Pearlman (8). The micro-Zimmcrmann reaction was iiscd in this assay (Figure 4, right). The product obtainrd shows excellent correlation with rommerrial proges- terone. The progesterone content has been determined using a plot of pro- gesterone concentration us. absorbance at 380 mp at a n established time; such a calculation showed tha t the placental sample contained 60 pg.

The Etudy was extended to inclnde LITERATURE CITED

the reaction" with the rwgcnts as mod- ified by Rlcndshoj whkh employs methanolic KOH. The proccdure wax investigated to test the practicability of simplifying the proredure for routine use, since methanolic KOH can be kept indefinitcly (4) . Figure 5 (left) is a repetitive scan of compound F, which is an unsaturated A'-3,20- ketosteroid. The composite picture shows thnt peaks develop at 380 and 490 mp, indicating the presrnrr of 3- and 20-keto groiips. The compound F spectrum diffcrs from progrsterone in having a distinct 490-rnp peak.

Figure 5 (right) is a reaction carried out with the Klendshoj rengcnts in an attempt to characterize the type of ketosteroid in an adrenal cortical ex- tract. The interesting aspect of this procedure is that by using a simple solvent extraction a peak absorbance occurs at 490 mu. indicatine: the Dres-

(1) Callow N. H., Callow, R. IC., Em- mens, 6. W., Biochem. J . 32, 1312 (1938).

(2) I'foltorff, A. F., Koch, F. C., J. Riol. Chem. 135, 377 (1940).

(3) Kaziro, K., Shimada, T., 2. physiol. Chen. 249, 220 (1937).

(4) Klendshoj, N., Feldstein, M., Spmpe, A.. J . Clin. Endoctinol. and ilfclabolism 13; 922 (1953).

BtoZ. Chem. 128,759 (1939). (5) Langst,roth, G. O., Talbot, N . J,., J .

(6) Marlow, H. W., Ibid 182, 167 (1950). 171 Nathnnson. I. T.. &Ison. H.. Endo- , , , ,

crinolo y 33,'189 (l???). (8) Pearfman, W. H. Rcccnt Progrcse in

Hormone ReRearch," Vol. IV, p. 27, Academic PresR, New York, 1954.

19) Strickler. H. S.. Wnlton. M. E.. Wil- ~, son, D. A:, Dienes, M., Endocrinology 29, 545 (1941).

(10) Wilson, H., Arch. Biochem. Riophys, 52, 217 (1954).

(11) Zimmermann, W., 2. physioi. Chem. 233. 257 11936).

(12) h i d . , 3 0 0 ~ 1 4 1 (1955).

ence of a 20 ketonic groupinichara'cter- istic of some corticosteroids.

R E c ~ ~ E ~ f o r r e v i e w January 3 1961. Ac- cepted March 29, 1961. Pittsturgh Con- ference on Analytical Chemistry and A died Snectrosconv. Pittsburah. Pa.. Fe&

ence of a 20 ketonic groupinichara'cter- istic of some corticosteroids.

R E c ~ ~ E ~ f o r r e v i e w January 3 1961. Ac- cepted March 29, 1961. Pittsturgh Con- ference on Analytical Chemistrv and AD- died SDectrosco-nv. Pittsburah, Pa.. Feb-

A C K N O W L E D G M E N T hary-March l96b: Work Su orted in part by grants-in-aid from the K a l t h Re-

Unitfed Fund of Allegheny County (B-1) and the Nationa] Institutes of IIealth (A 3689).

The authors thank Robert K ~ ~ ~ ~ , search and SclViCeS Foundation Of the Fisher Scientific Co., for wiring the timer into the circuit of the Reckman DK-1.

Spectrometric Determination of 9 Alpha-Fluorohydro- cortisone in Fermentation Broths

EUGENE IVASHKIV

Squibb lnsfitute for Medical Research, New Brunswick, N. .I.

b A rapid chemical method has been devised for determining 9a-fluoro- hydrocortisone in the presence of 9a- fluoro-l6a-hydroxyhydrocortisone in fermentation broths. The method de- pends upon the formation of a pink color from 9a-fluorohydrocartisone in the presence of cysteine, fructose, and sulfuric acid. Concentrations as low as 5 pg. per ml. of 9a-fluoro- hydrocortisone can b e determined.

UMEROUB methods have bccn pub- N lished on the chemical assay of 17-hydroxycorticosteroids and related compounds. Many methods of puri- fication of steroid extracts have been reported. All these methods have been reviewed recently (I), but none of them could be applied to fermentation process control in the conversion of 9a-fluorohydrocortisone to 9a-fluoro- 16a-hydroxyhydrocortisone. Recently, Sih, Pan, and Bennett of these labora- tories proposed a colorimetric method

(9) for determination of Sa-fluoropred- nisolone and Sa-fluorohydrocortisone in triamcinolone samples by employ- ing aqueous solutions of fructose, cys- teine, and sulfuric acid.

To adapt this method to meet the requirements of process control in the conversion of Sa-fluorohydrocortisone to 9a-fluoro-l6c~-hydroxyhydrocortisone several modifications of the pro- posed method have been made: The water content in the reaction me- dium is kept a t a minimum by using methanolic reagents; concentrations of fructose and cysteine are decreased considerably; the reaction is carried out at 100' C. and in the absence of atmospheric oxygen: after the reaction is completed, interfering colors are eliminated with addition of distilled water; a method for extraction of steroids from fermentation broths and the removal of interfering substances with buffer solution and methylene chloride is used; and turbidity in eamples, caused by defoaming agents

used in the fermentation procesg, is removed b y extraction with diethyl ether.

METHOD

Reagents and Equipment. CYS- TEINE HYDROCHLORIDE-FRIJCTOSB SOLUTION. One gram of I-cysteine hydrochloride is dissolved in about 100 ml. of methanol, 2 ml. of 0.1% methanolic fructose solution (0.100 gram per 100 ml.) is added, and the volume is made u p to 200 ml. with methanol.

METHYLENE CHLORIDE (DICHLORO- METHANE). Reagent suitable for spec- trophotometric use.

BUFFER SOLUTION, pH 10.5. Eighty grams of sodium bicarbonate, 50 grams of sodium chloride, and 453.6 grams of anhydrous potassium carbonate a re dissolved in distilled water to obtain the final volume of 1000 ml.

STANDARD %-FLUOROHYDROCORTI- SONE SOLUTIONS. Sa-Fluorohydro- cortisone standard is dissolved in methylene chloride to give the final

VOL. 33, NO. 8, JULY 1961 0 1051

0.30

E 3

0.20 0

0.10

/ 0.3

. Opened Test Tubes I. Sloppered T e d Tubes A - Tubes Under N2

I

60 I20 I80 240 300 Concentration, ,ug./cc.

Figure 1. Effect of oxygen on the reaction at 100' C.

concentrations of 10, 20, and 40 p g . per ml.

TEST TUBES, screw cap. 25 X 150 mm. and 20 X 150 mm. with cork- backed vinylite caps.

NITROGEN SOURCE. Nitrogen cylin- der equipped with valves and a niani- fold.

PROCEDURE

Extraction. A 3-ml. broth sample is transferred into a test tube con- taining 0.5 gram of sodium bicarbon- ate, 15.0 ml. of methylene chloride is added, and the tube is covered with a plastic cap. The steroids arc ex- tracted b gentle shRking for 1 minute. *he sample i s centrifuged a t 1000 r.p.m. for 1 minute. The upper, aqueous layer is discarded by means of an aspirator. Ten milliliters of the methylene chloride extract is transferred into a clean 25 X 150 ml. test tube, 10 ml. of buffer solution is added, and the tube is shaken vigorously for exactly 10 seconds. The solvent and the aqueous layers are separated by centrifugation.

Blanks are prepared for each fer- mentation batch by removing a sample of broth prior to the addition of steroids. Then 30 ml. of this broth is transferred into a 250-ml. ccntrifuge tube contain- ing 5 grams of sodium bicarbonate, 150 ml. of methylene chloride is added, and the sample is shaken gently for 1 minute. After centrifugation, the aqucous layer is discarded, and the methylene chloride solution is refrigerated in a brown bottle. A 10-ml. aliquot of the blank solution is washed with t h c buffer just before the analysis of samples.

Color Development. I n fivv tcst tubes marked A , I3, 10, 20, and 40, 5 ml. of the methylene chloride sample extract, 5 ml. of thc blank extract, and 5 ml. of standard solutions a t the concentration of 10, 20, and 40 fig. per ml. are placed, respectively. To each tube 5 ml. of the sulfuric acid is added. The tubes are covered with plastic caps and shaken for 1 minute. The two layers arc alloivcd to separate com- pletely and the methylene chloride layer

0.2

:: r f f 4

0.11

(

Curves at Temperalurcs:

4

IO 20 30 40 50 60 70 80 90 Time, minutes

Figure 2. Effects of temperature and time on reaction rate

is discarded by means of an aspirator. Acid layers of the sample and of the blank are washed twice with 10 ml. of methylene chloride and after the separa- tion of two layers, the solvent layer is discarded. To the acid layer 1 ml. of absolute ethyl alcohol and 1 ml. of cysteine-fructose are added. All tubes are flushed well with nitrogen, covered with plastic caps, mixed, placed in a boiling water bath, and heated for exactly 12 minutes. The test tubes are cooled in an ice bath, and 4 ml. of dis- tilled water i s added and carefully mixed. The color is stable for several hours. If a brown color is observed in a sample or in a blank, i t can be removed by extraction with methylene chloride; defoaming agents causing turbidity are removed by extraction with 15 ml. of absolute dicthyl ether. The ether layer is discarded by means of an aspirator. Ethcr left in the acid layer is removed by bubbling nitrogen gas into the sample. The absorbance of the blank, of thr sample, and of the standards is measured a t 546 mfi on the spectro- photometer using 1-cm. silica cells and 85% sulfuric acid for the adjustment of the instrument to 100~o transmittance.

The amount of Sa-fluorohydrocorti- sonr is calculated as follows:

( A x - K 'I5 = pg./ml. of broth

where A X = absorbance of the sample A B = absorbmce of the blank

and K = absorbance per fig. of 9a-

fluorohydrocortisone per ml. of standard in methylene chloride.

DISCUSSION

Methyl isobutyl ketone, a good sol- vent for thc cxtraction of steroids, was unsuitable for this method, as i t gave the same color with the reagents as did Sa-fluorohydrocortisone. When methylene chloride was used for ex- traction, this interference was not ob-

served. By employing 15 ml. of meth- ylene chloride as solvent, 97 to 99% of the steroids were removed from 3 ml. of fermentation broth by one extraction.

Standards of 9,-fluorohydrocortisone were prepared in dimethylformamide; 0.3-ml. aliquots of these standard solutions were added to 3-ml. portions of a fermentation media and the steroid was extracted with 15 ml. of methylene chloride. The recovery of Sa-fluoro- hydrocortisone is tabulated in Table I.

Table 1.

Steroid Added r d M f .

15 15 30 50 100 150 200

Recovery of 9a-Fluorohydro- cortisone Batch No.

1 2 3 Av. Re- Steroid Recovered, covery,

rg./M1. %

15.0 13.9 15.6 98.9 28.9 31.7 28.3 98.8 48.5 49.0 49.6 98.1 98.1 98.7 98.1 98.3 150 150 148 99.6 198 200 200 99.7

A study of the stability of 9a- fluorohydrocortisone in methylene chlo- ride solution determined by Blue Tetra- zolium and paper chromatographic methods showed that the steroid is stable for several days if kept re- frigerated in a dark bottle.

An increase in the concentration of sulfuric acid increases the rate of re- action but high concentration leads to undesirable by-products and lowering of absorbance. Data indicate that a4 least 70% sulfuric acid is needed for the reaction, and when the concentration of the acid falls below 60% reaction almost ceases.

The concentration of cysteine- fructose is very important in color development. Either cysteine or fruc- tose produces a pink color, but employ-

1052 * ANALYTICAL CHEMISTRY

rnent of both reagents gives a higher color intensity. i\ eoncentration of 0.006% fructose results in a pink- purple color m d when the concentra- tion is increased to 0.1% or more a brown color is obtained. Cysteine a t concentrations of 3% and higher pro- duces pink-purple and purple colors. A combination of 0.5yo cysteine and 0.002yo fructose as the reagent for carrying out the reaction proved to be most suitable.

In an effort to decwase the reaction time to a minimum by raising the tem- perature, the effect of oxygen on the reaction was studied. When the re- action was carried nut at elevated tempcrat,ures to accelerate the rate and degree of the rca,ction, the color dis- appcarcd.

The effect of osygcn on the reaction a t high temperatures is shown in Figure 1. 'I'riplicat,e samples were prepared at diffcrent concentrations and were treated as follows: The first set of samples was heated in ope11 test tubes, the sccond sct of snniplcs was stoppered with riibhrr stoppcrs, and the third set was heated under a stream of nit,ro- gcn. In thc second sct of samples a dwreasc in volume and, therefore, a dwreasc in prcxjure were noticed, for it was d i f f i d t to rcinovc the rubber stoppers from thc test tubes. This

during the reaction m d this resulted in a part,ial disappearance of the color.

suggestcd that oxygm was used up

Table II. Reproducibility of Results

F ~ ~ ~ ~ ~ - Concn. of Unconverted tation 9a-Fluorohydrocorti-

sone, pg./Ml. Time, __ Hours Sample 1 Sample 2 Range

1 292 295 3 .O 43 195 191 4 0

117 0 0 . . . 122 0 0 . I .

Heating the samples under a stream of nitrogen protected the reaction mix- ture during the process.

The most important factor influenc- ing the velocity of the reaction was the temperature. An increase in tem- perature accelerates both the rate and degree of reaction. The reaction was carried out at room temperature (about 30" C.), 40°, 70°, and 100" C.

Figure 2 illustrates the relationship between temperature and time of heat- ing necessary to reach equilibrium. The equilibrium state is reached in about 130 hours a t room temperature but in 1 1 minutes a t 100" C. At tem- peraturrs of 40" and 70" C. ' a state of equilibrium was obtained in about 55 minutes but with a very low Golor de- velopment. The necessity for com- pleting the test in a minimum of time led to the use of a high tcmperature to

increase the speed of the rraction. Substances used in the preparation of a fermentation media contribut,c to the color developed in fermentation ex- tracts. These substances can be eliminated from a sample by washing the methylene chloride sample with buffer solution. Washing the methylene chlorido extract with the buffer for 10 seconds resulted in a 0.5 to 1.0% loss of steroid into the buffer layer. The loss increased up to 12% after the 5- minute washing. In samples taken dur- ing the latter stages of fermentation a brown color sometimes interferes in the determination of absorbance. This interference can be eliminated by ex- tracting the steroid into the acid layer and washing off the impurities with methylene chloride.

T o check the reproducibility of results, analyses of several fermenta- tions were performed in duplicate. Results of a typical run are given in Table 11.

The data indicate that results can be reproduced within *l'% at higher concentrations, and at lower conccntra- tions the results differ by *3.0y0 from the average.

(1) Rehm G . R., Slack, S. C., Mnder, W. J., ANAL. CHEM. 31, 749-66 (1959).

(2 ) Sih, C. J., Pan, S. C., Bennct.t, R. E., Ibid., 32, 669 (1960).

RECEIVED for review January 81, 1961. Accepted May 9, 1961.

LITERATURE CITED

Microdetermination of Acetals of Acetaldehyde, Vinyl Ethers, and Other Compounds Containing Corn bined Acetaldehyde Groups

MALCOLM C. BOWMAN,' MORTON BEROZA, and FRED ACREE, Jr.

Enfomology Research Division, U. S. Department of Agriculture, Orlando, Ha., and Beltsville, Md.

b A method has been developed for determination of the pyrethrin and carbamate synergist, sesamex, which is also useful for determination of other compounds that liberate acetaldehyde on acid hydrolysis. The compound i s hydrolyzed with sulfuric acid, and the acetaldehyde, after steam-distilling, is treated with a solution of p-phenyl- phenol and cupric sulfate in concen- trated sulfuric acid to give a violet color absorbing at 572 mp. N o interference from the insecticides syn- ergized by sesamex was encountered. The method is potentially useful for the structure determination of com- bined acetaldehyde groups in minute amounts of compound.

HE analytical method described T here stemmed from a desire to deter- mine micro Rmounts of scsamex ( 4 ) [Sesoxane, 2-(2+thoxycthoxy)ethyl 3,4- methylenrdioxyphrnyl acetal of acetal- dehyde] in mi\tures with the insecti- cides it syncrgizes. This method ap- pears to be gericrally useful for the de- termination of compounds containing combined acet:iltlchyde groups that can be liheratcd by trcntrnent with an aque- ous mincrnl :~cicl. Examples are ace- tals of ac~tnldchydr, ncetaldrhydc poly- mers, and vinyl ethers. Vinyl cthcrs have recmtly betn made available ?om- mercially and products, like sesamex,

' I'resent address, Campbell Soup Co., Camden, N J.

based on vinyl ethers are being de- veloped and may be amenable to the prcsent method of analysis.

The method is a modification of the one employed by Barker and Summer- son (1) for lactic acid in biological ma- terials, by Stotz (13) for acetaldehyde in blood, and by Giang and Smith (9) for metaldehyde (tetramer of acetaldehyde) in plant material. The compound is hydrolyzed with sulfuric acid; the acetaldehyde generated is distilled into aqueous sodium bisulfite and reacted with p-phenylphcnol in the presence of concentrated sulfuric acid and cupric ion. A violet color having an absorp- tion maximum a t 572 mw (Figure 1) is produced.

VOL. 33, NO. 8, JULY 1961 1053