ceivable that in sampling air nearly saturated with water vapor, the silica gel might become inactive after exposure to a relatively small sample. This can be eliminated by the inclusion of a trap cooled to dry ice temperature before the detector tube. Substances which form colored compounds with the reagent are more difficult to remove. Any sub- stance having a negligible vapor pressure a t -78” C. can be removed in the U-tube trap, or, if present in sufficiently small quantity, by the glass beads in the de- tector tube preceding the gel layer. Un- fortunately, the substances most likely to interfere are not completely removed by cooling to -78” C., because their vapor pressures a t this temperature are too high.

The test described is not specific for acetylene, but is one for any alkyne in which the triple bond occurs a t the end of a chain. High molecular weight alkynes with low vapor pressures can be removed as easily as water. For al- kynes from 1-propyne to pentyne, the vapor pressures a t dry ice temperature are such that little if any would be re- tained in the trap. However, it is im- probable that these alkynes would be found a t the same place and a t the same concentration as acetylene, whether the source of the sample were an urban at- mosphere, or an industrial area produc- ing and using acetylene, where the acety- lene would be the primary alkyne lost to the atmosphere. Other compounds

which have relatively high vapor pres- sures and interfere are hydrogen sulfide and some mercaptans. Hydrogen sul- fide precipitates black cuprous sulfide from an ammoniacal cuprous solution. Mercaptans precipitate yellow salts that may be mixed with copper sulfide (7) and either rvould modify the color pro- duced by the acetylide if present in appreciable quantities. The presence of the low boiling mercaptans a t concentra- tions of parts per billion is evident from their odors while hydrogen sulfide is said to have a detectable odor a t a concentra- tion as low as 0.75 p.p.m. (5 ) . There- fore, either substance would be noticed before it constituted a serious inter- ference.

CONCLUSION

The method can be used to estimste the amount of acetylene in air a t con- centrations as lonr as 0.001 p.p.m. Quantitative results are easily obtained in the range from 0.1 to 10 p.p.m. A single gas mixture having a concentia- tion of acetylene of about 1 p.p.m. is sufficient for a calibration in this range. It is necessary to cool the collecting media to dry ice temperature when de- termining concentrations below 10 p p.m. Numerous determinations can be made rapidly and easily. The qualitative detection of acetylene in the range from 1 p.p.b. to 0.1 p.p.m. is possible, provided more care and greater

volumes of saiiiple are employed. It is probable that by reducing the tempera- ture of collection and increasing the to- tal voluine of sample, the quantitative determination can be extended to below 1 p.p.b.

The method can be used in the field almost as easily as in the laboratory. Tubes can be exposed in the field, stored in liquid nitrogen, and returned to a more convenient location for develop- ment and comparison. If tubes can be returned to the laboratory within 30 minutes, storage in dry ice is permissible. Because of the simplicity of the pro- cedure for color development and the small amount of reagent required, tubes may be compared a t the sampling site with standard tubes.

LITERATURE CITED

(1) Berthelot, A f . , Compt. rend. 54, 1070

( 2 ) Feigl, Fritz, “Spot Tests in Organic Analysis,” 5th ed., pp. 323-4, Van Sostrand. Kew York. 1956.

(1862).

(3) Ilosvay,’Ludwig, Be?. 32; 2697 (1899). (4) Purser, B. J., Analys t 78, 732 (1953). ( 5 ) Sayers, R. R., Dalla Valle, J. M.,

\-ant. T T . P.. Ind. Ena. Chem. 26, 1251 (1934).

(6) Shepherd, Martin, AXAL. CHEJZ. 19, 77 (1947). - \ - - - ,.

( i ) Veldheer, P. A, Chem. TYeekblad 44, 499 (1948).

RECEIVED for review March 22, 1958. Ac- cepted August 27, 1958.

De te rm i n a t i o n of A 5 - 3 - Bet a - H y d r oxys te ro i ds GEORG W. OERTEL and KRISTEN B. EIK-NES Deparfment o f Biochemistry, College of Medicine, University of Utah, Salt lake City, Utah

Less than 1 y of A5-3/3-hydroxy- steroids can be determined by reaction with a sulfuric acid-ethyl alcohol re- agent. A yellow color is developed within 2 minutes and is stable for at least 2 hours which has an absorption maximum at 400 to 405 mp. This re- action may be used for the quantita- tive determination of these steroids in biological fluids.

OST of the procedures for the estimation of As-androstene-3/3-

01-17-one (dehydroepiandrosterone) and other 3p-substituted 17-ketosteroids ( I , 2, 4 ) require a t least 10 y of steroids for an adequate detection, a concentration rarely present in physiological amounts of blood plasma. A reaction is re- ported which allows the determination

of 0.5 y of a A5-3p-hydroxysteroid with fair accuracy.

EXPERIMENTAL

From 0.5 to 20 y of any A5-3p- hydroxysteroid are dissolved in 2.5 ml. of reagent, prepared from 1 volume of 95% ethyl alcohol (freshly distilled in vacuo over silver oxide) and 2 volumes of concentrated sulfuric acid (analytical grade). After 2 to 15 minutes a t room temperature, the reaction mixture is transferred to a 3-mi. cuvette and the absorption spectrum of the solution is registered in a Beckman DK 2 recording spectrophotometer betlveen 350 and 500 mp; a reagent blank is used as reference. Ki th the exception of de- hydroepiandrosterone, which exhibits an absorption maximum a t 400 mp, a peak absorbance is observed a t 405 mp for the A~-3p-hydroxysteroids tested. Because the absorption curves of the reagent mixture, as well as those of

contaminating material which can be eluted from paper chromatograms with methanol, follow a straight line be- tween 370 and 430 mp, the maximum absorbance a t 405 mp may be corrected by the application of a formula similar to that developed by -4llen (1 ) :

Corrected absorbance at 405 mp = ob- served absorbance at 405 mp less one half the sum of absorbance at 380 and 430 mp

In the case of dehydroepiandrosterone, ware lengths 375, 400, and 425 mp are selected for the correction of the maximum absorbance a t 400 mp.

RESULTS

The absorption spectra obtained with 0.5, 1, 2, 3, 4, 5, and 10 y of As- pregnene-3p-ol-20-one (pregnenolone) are shown in Figure 1. The values for the corrected absorbance a t 405 mp are listed in Table I.

98 ANALYTICAL CHEMISTRY

The use of 1.5-ml. microcuvettes increases the sensitivity of the proce- dure, allowing the estimation of about 0.1 y of 46-3P-hydroxysteroids (Figure 2 ) .

To follow the development of color with time, the absorption spectrum of 10 y of pregnenolone in 2.5 ml. of rea- gent was determined a t various time intervals. Within 120 seconds this reaction reaches equilibrium and after 120 minutes shows significant fading. The sensitivity of the method depends on the ratio of the relative concentration of ethyl alcohol and sulfuric acid (Table 11).

A mixture of 10 y of androstane-3p- ol-17-one and 10 y of dehydroepian- drosterone gave a corrected absorbance for dehydroepiandrosterone of 0.0427 per microgram as compared to 0.0439 per microgram for pure standard de- hydroepiandrosterone, indicating that the presence of a steroid with a con- figuration other than A5-3p-hydrosy did not influence the color formation, This experiment was done using 1.1 nil. of reagent.

To check precision, 8 samples of 10 y of 4s-pregnenolone each were as- sayed, using 2.5 ml. of reagents. The average corrected absorbance per micro- gram w s 0.0189 =+= 0.0010 (standard deviation).

Ten micrograms of various A5-39- hydroxysteroids were subjected to the procedure (Table 111) and the corrected absorbance was calculated. In the case of dehydroepiandrosterone, the ab- sorbances a t 375, 400, and -125 mp were used for calculation. All of these A5-3p-hydroxy compounds gave a maui- mum absorbance betv-een 400 and 405 mp. However, the functional groups in the steroid molecule and the spacial Configuration of the molecule seem t o influence the extinction.

Androstane-3 a-01- 17-one, androstane- 3p-ol-li-one, etiocholane-3cr-ol-17-one, etiocliolane-3~-ol-17-one, A4-androstene- 3,17 - dione, A4 - androstene - 118-01- 3,17-dione, A4-androstene-17~-ol-3-one, pregnane - 3cu,2Oa - diol, A4 - pregnene- 21-01-3,20-dione, A4-pregnene-11p.21- diol-3,20-dione, A4-pregnene-1 13,1 io(, 21-triol-3,20-dione, allopregnane-38, ll~,17cu.21-tetrol-2O-one, and A4-preg- nene-3,20-dione were assayed under the same conditions, but did not exhibit an absorption masimum between 380 and 430 nip.

DISCUSSION

Zaffaroni (6) has described the chro- mogen formation of adrenal steroids and related compounds with concen- trated sulfuric acid. Dilution with methanol of the yellow color produced will shift the absorbance to the red region of the spectrum (3) where less back- ground absorbance occurs. In Figure

'70r A

366370 380 390 400 425 4 5 0 WAVE LENGTH IN MILLIMICRONS

Figure 1. Absorption curves of 0.5, 1, 2, 3, 4, 5, and 10 y of As-pregnene- 3P-ol-20-one in 2.5 ml. of reagent

WAVE LENGTH IN MILLIMICRONS

Figure 2. Absorption curvesof 0.1,0.5, 1 , and 10 y of A5-pregnene-3p-01-20- one in 1.1 ml. of reagent, read in micro- cuvette of 1.5-ml. capacity

*

I 1 I 1 425 450 366371.1 380 390 4 0 0

IVAVE LENGTH IN MILLIMICRONS

Figure 3. Absorption curves

a. concentrated sulfuric acid (5 ) b. reagent

10 y of A5-pregnene-3P-oC20-one in 1 ml. of

1 y of 46-pregnene-3,B-ol-20-one in 1 ml. of

3 are recorded the Chromogen spectrum of 10 y of pregnenolone dissolved in 1.1 ml. of concentrated sulfuric acid and processed as outlined by Zaffaroni, and the chromogen curve of 1 y of preg- nenolone dissolved in 1.1 ml. of 95% ethyl alcohol-concentrated sulfuric acid 1 to 2. The latter sample was read 15 minutes after the addition of the rea- gent, and a quartz fused cell with a

Table I. Corrected Absorbance ( 7 ) a t 405 Mp for Increasing Amounts of Pregnenolone in 2.5 MI. of Reagent

(Data compiled from Figure 1) ilbsorb-

Pregneno- Corrected ance/y lone, Absorbance Preg-

-/ at 405 hfp nenolone 0 . 5 0.009 0.0180 1 0.018 0.0180 2 0.037 0 . 0 1 85 3 0.052 0 . 0i73 4 0.073 0.0183 5 0.095 0.0196

10 0.191 0.0191 20 0.366 0.0183 AI-. absorbance per y . 0.0184 5 0.007

Table I I . Corrected Absorbance ( 1 ) for 10 y of Pregnenolone in 2.5 MI. of

Reagent of Varying Composition (1 volume of ethyl alcohol)

Volume Ethyl of Corrected

Alcohol. Sulfuric Absorbance Concn., % Acid at 405 hIp

80 1 0.031 2 0.160 3 0.153 4 0.060

90 1 0.123 2 0.173 3 0.158 4 0.050

100 1 0.178 2 0.172 3 0.125 4 0,076

Table 111. Corrected Absorbance ( 7 ) a t 405 Mp (400 Mp) of 10 y of Various A5-3/3-Hydroxysteroids in 2.5 MI. of

Reagent Corrected Absorb-

ance rtt 4

Compound hIp A5-dndrostene-3p-ol-17-one 0 186 As-Androstene-3p- 17p-diol 0 146 A5--4ndrostene-3p, 17a-diol 0.162

A6-16-Pregnadiene-3p-ol-20-one 0 258 A5--Androstene-3p, 16p-diol 0 110

total capacity of 1.5 nil. was used for both samples. Apparently, this is a different type of chromogen formation in the reaction than that found by Zaffaroni, or else the sensitivity of the original Zaffaroni reaction has been increased.

To determine the application of this method the estimation of A5-3p- hydroxysteroids in biological material, 1, 2, 5, and 10 y of pregnenolone were added to 10 ml. of artificial human plasma and recovered according to a procedure similar to that used for de- termination of dehydroepiandrosterone in plasma ( 5 ) ; 0.76, 1.59, 4.13, and 8.12

VOL. 31, NO. 1, JANUARY 1 9 5 9 99

Y of pregnenolone were detected. This Morris and Renate Oertel for their color reaction can be used for the technical assistance. estimation of dehvdroetiandrosterone in human urine and plasma, if extracts of these fluids are purified by paper chro- LITERATURE CITED

~~

matography (6). (1) Allen, W. M., J . Clin. Endocrinol. 10, 71 (1950).

(2) Dhscherl, Wilhelm, Zilliken, Friedrich, Naturwissenschuflen 31, 349 (1943).

(3) Linford, J. H., Can. J . Biochem. and ACKNOWLEDGMENT

The authors wish to thank Mavis Physiol. 34, 1153 (1956).

Determination of Novobiocin in the Presence of Isonovobiocin

ARLINGTON A. FORIST, SUSAN THEAL, and WILLIAM A. STRUCK Department o f Physical and Analytical Chemistry,, The Upjohn Co., Kalamazoo, Mich.

b Novobiocin frequently contains varying amounts of isonovobiocin, which is devoid of antibiotic activity. The two isomers are indistinguishable by physical measurements. A chemical procedure for the determination of novobiocin in the presence of isonovo- biocin is based on cleavage of the novobiocins with anhydrous trifluoro- acetic acid to yield the respective 0- carbamylnovioses, followed by deter- mination of periodate consumption by 3 -0-carba mylnoviose from novobiocin. 2-0-Carbamylnoviose from isonovo- biocin does not interfere. Analysis of standard novobiocin samples indicates excellent accuracy and precision (mean recovery f standard deviation 99.9 f 1.5%). Mean deviation for novo- biocin in samples containing 0 to 20y0 isonovobiocin is f 1.5%.

HE ANTIBIOTIC novobiocin has been T assigned structure I (3, 6-8, 10-12). Hinman, Caron, and Hoeksema (4) have reported the isomerization of I to give isonovobiocin (11) by a carbamyl migration. Because I1 is inactive as an antibiotic, an analytical procedure is needed which will permit the deter- mination of I in the presence of I1 with- out disturbing the ratio of the two forms,

The two isomers are indistinguishable by ultraviolet or rotational measure- ments and shorn only slight differences in their infrared spectra. Published methods for the analysis of mixtures of I and dihydronovobiocin (9) and for the determination of 1 in plasma and serum (1) do not differentiate I and 11. The procedure reported here permits the direct determination of I in the presence of 11. It is based on the perio- date oxidation of 3-0-carbamylnoviose produced from I by acid cleavage of the glycosidic bond.

REAGENTS

Trifluoroacetic Acid, anhydrous. Sodium Arsenite, 0.1000N. A sample

of 9.8910 grams of primary standard arsenious oxide is added to a 2-liter volumetric flask containing 20 grams of sodium hydroxide in 50 ml. of water. The solution is diluted with about 300 ml. of water and 42 ml. of concentrated hydrochloric acid are added, followed by 20 grams of sodium bicarbonate. The resulting solution is diluted to 2 liters.

Periodic Acid, 0.2N. Twenty-three grams of o-periodic acid are dissolved and diluted t o 1 liter with water. This solution is stored in an amber bottle and standardized by the following modifica- tion of the method of Fleury and Lange (2 ) . Duplicate 4.00-ml. aliquots are saturated with sodium bicarbonate. A 10.00-ml. aliquot of the 0.1000N sodium arsenite solution is added to each, fol- lowed by 2 ml. of 5% potassium iodide. After a t least 15 minutes, the excess arsenite is titrated to a starch end point with the standard 0.0lhr iodine solution. Additional sodium bicarbonate may be required during the titrations to keep the solutions saturated (solid phase present). Weekly restandardization is recommended.

Iodine, Stock Solution, 0.1N. A mix- ture of 12.7 grams of iodine and 60

(4) Munson, P. L., Jones, 31. E., McCall, P. J., Gallagher, T. F., J . Biol. Chem 176, 73 (1948).

( 5 ) Oertel, G. W., Eik-Kea, K. B., Ibid., in press, 1958.

16) Zaffaroni. Aleiandro. J . Am. Chem. . , SOC. 72, 3828 (1950).

RECEIVED for review February 20, 1958. Accepted August 4, 1968. Work sup- ported by United States Public Health Grant CRTY-5000 Xational Institutes of Health, Bethesda 14, Md,

grams of potassium iodide is dissolved in 75 ml. of water and diluted to 1 liter. This solution is stored in a tightly stop- pered bottle protected from light.

Iodine, Working Solution, 0.01N. This is prepared by a tenfold dilution of the stock solution and is stored in a tightly stoppered bottle protected from light. This solution is standardized daily by the titration of 3.00-ml. ali- quots of the 0.1000N sodium arsenite reagent, saturated with sodium bicar- bonate, to a starch end point.

Potassium Iodide, 5%. Five grams of potassium iodide are dissolved in 100 ml. of water and this solution is satu- rated with sodium bicarbonate. The re- agent prepared in this manner is stable for several weeks.

PROCEDURE

Glycoside Cleavage. d sample of 40 to 50 mg. of the material to be analyzed is accurately weighed into a 125-ml. glass-stoppered Erlenmeyer flask or alternatively, into a 50-ml. glass-stoppered centrifuge tube. Five milliliters of anhydrous trifluoroacetic acid are added to the sample, the vessel is tightly stoppered, and the resulting solution is allowed to stand a t room temperature in the dark. At the end of 2 hours, 20.00 ml. of water are slowly added with swirling. The precipitate which forms is separated

a short centrifugation, followed by 2 ration of the supernatant through a medium porosity glass-fritted funnel under vacuum. The vacuum is released as soon as the filtration is complete to avoid loss of solvent by evaporation. The resulting filtrate contains the 0- carbamylnovioses.

Accurately weighed samples of ap- proximately 20, 40, and 60 mg. of a standard novobiocin preparation are carried through the procedure above in parallel with the unknown.

Periodic Acid Oxidation. A 20.00- ml. aliquot of each aqueous trifluoro- acetic acid filtrate is transferred to a

100 ANALYTICAL CHEMISTRY