proximately 1 ml. of sodium cupribromide reagent, stopper, and shake. A violet to blue color that develops after a few minutes in the supernatant layer is a positive test for 1-naphthol. This procedure can be used for semiquantitatire work by calibrating the scoop and comparing the developed color with standard color tubes. Quantitative Method. Weigh a 0.500-gram sample of napalm into a 50-ml. glass-stoppered Erlenmeyer flask. Add 25 ml. of mixed solvent and shake until the napalm is in solution or evenly suspended in the solvent. As a reagent blank, treat a sample of approximately 0.5 gram of 1-naphthol-free napalm in the same manner. Add 1 ml. of sodium cupribromide reagent to each, shake, and filter through a No. 4 Whatman filter paper into a calibrated Klett tube to the 5-ml. mark. Dilute to 10 ml. mix., and measure the absorbance using filter Xo. 54. Make the measurements within 5 minutes after the original mixing with reagent. Calibration. Prepare 1-naphthol standards containing 10 t o 50 y per ml. in the mixed solvent. Add 25 ml. of each to 50-ml. glass-stoppered Erlenmeyer flasks containing 0.5 gram of 1naphthol-free napalm. Proceed as for the sample determinations and prepare a calibration curve of instrument readings us. concentration. RESULTS
Synthetic samples were prepared by adding known quantities of 1-naphthol to oxidation inhibitor-free napalm. Other specimens were prepared by adding excesses of oleic acid (20%) and naphthenic acids (127,) over and
beyond that already present in the napalm. KO interference was encountered and recoveries of 1-naphthol within 2% were obtained (Table I).
Table II.
color or extremely poor sensitivity. Recoveries within 2% mere obtained for 1-naphthol when determined in mixtures containing the above compounds a t 10 times the weight of the 1-naphthol.
Tests of General Application
Phenol 2-Naphthol I-Naphthol-2-sulfonic acid 2,7-Dihydroxynaphthalene I,1’-Bi-2-naphthol Catechol 1,GDihydroxynaphthalene-3,6-disulfonic acid
Reaction with Cupribromide Pale yellow Yellow Yellow Yellow Yellow Brown No color
General Application of Method. The sodium cupribromide reagent was added to known mixtures composed of microgram quantities of 1-naphthol and milligram quantities of other phenols. The mixtures were dissolved in 50% acetone-water solution. Of the phenols tested only catechol showed color development other than yellow and that with poor sensitivity (Table 11). I n this case the brown color could be differentiated spectrophotometrically from the blue of the 1-naphthol complex. Phenol, 1,8-dihydroxynaphthalene-3,6disulfonic acid, 2-naphthol. l-naphthol2-sulfonic acid, 2,7-dihydroxynaphthalene, l,5-dihydroxynaphthalene.and l.l’-bi-2-naphthol shoxed either little
CONCLUSIONS
A little used reaction for the differentiation of 1- and 2-naphthol and some alkaloids from one another has been adapted to a quantitative colorimetric method. Although most of the work described in this report was done on a particular problem, sufficient tests were made to indicate the possibility of a more general usage of the method. The method can be made sensitive to 1 y of 1-naphthol per ml. of solution when used for the direct estimation of the compound in the presence of similar compounds. LITERATURE CITED
Fieser, L. F., Harris, G. C., others, Ind. Eng. Chem. 38, 768-73 (1946). Liebmann, J., J . SOC.Chem. Ind. 16, 294 (1897). Prochazka. J.. Ind. Ena. Chem. 15.944 (1923). Rosenthaler, L., Pharm. Acfa Helv. 13, 3 (1938). Sa, A., Marsico, D., Anales asoc. qutm. argenfina31, 202-12 (1943). White, E. P., ISD.ENG.CHEM.,ANAL. ED.13,509 (1941). I
,
RECEIVEDfor review May 9, 1956. Accepted August 21, 1956. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., February 27, 1956,
Evaluation of Silber-Porter Procedure for Determination of Plasma Hydrocortisone RALPH E. PETERSON, AURORA KARRER, and SERAFIM L. GUERRA National Institute o f Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Md.
Silber and Porter recently published a novel procedure for the determination of plasma hydrocortisone, which appeared to offer many advantages over previously published methods. Experimental data on various aspects of the procedure are presented here, including an evaluation of its specificity for plasma hydrocortisone. Simplifications of the method are also described.
T
HE isolation and identification of hydrocortisone in adrenal gland perfusates (9) and in human peripheral blood (6) have demonstrated that this
144
ANALYTICAL CHEMISTRY
steroid is one of the chief cortical steroids secreted by the adrenals in man. Until recently, the most widely used method for the determination of hydrocortisone in human peripheral blood has been that of Nelson and Samuels (6), utilizing a Florisil column for purification of the plasma extract and the Porter-Silber phenylhydrazinesulfuric acid-ethyl alcohol reagent ( 8 ) for colorimetric assay. This method, with minor modifications (2, 3, IS), has been used by several investigators to measure plasma hydrocortisone levels. Silber and Porter (IO) h a r e recently reported a much simpler method, which
also utilizes the Porter-Silber reagent. This method is based on the following principles: Hydrocortisone is extracted froni plasma with an organic solvent. The solvent is washed with aqueous alkali to remove a considerable amount of “blank” material. The steroid is extracted from the solvent with a sulfuric acid-aqueous ethyl alcohol reagent, containing phenylhydrazine. After the phenylhydrazine has reacted with the hydrocortisone, the resulting colored product is measured spectrophotonietrically a t 410 mp in the acid phase. A correction for material in plasma reacting with sulfuric acid is
made by treating a n equal aliquot of the plasma extract with sulfuric acidethyl alcohol which contains no phenylhydrazine. The following modifications of the original Silber-Porter method are presented: substitution of dichloromethane for chloroform and its purification by use of silica gel; extraction procedures that did not require centrifugation; the use of a smaller volume of phenylhydrazine reagent, vhich makes i t possible to use a smaller volume of plasma and also increases the sensitivity of the measurement; and incubation of the phenylhydrazine reagent a t room temperature.
I
1
I
I
I
Dichloromethane. This solvent (Eastman, c.P.) is purified by passing i t through a bed of silica gel (average 100 mesh) in a 7 x 130 cm. column and collecting %liter portions in separate containers. Ten t o 30 liters can be purified in 2 t o 3 hours. T h e effectiveness of purification is determined by shaking a 20-ml. aliquot of solvent with 0.5 nil. of phenylhydrazine-sulfuric acid-ethyl alcohol reagent. X o color should develop on standing overnight a t room temperature. The solvent has remained free of impurities for many months a t room temperature. Sodium Hydroxide, 0.1N. Sulfuric Acid, 64y0. C.P. grade sulfuric acid (640 ml.) is added to 360 ml. of water. T h e flask should be kept cool under a running cold mater t a p during addition of sulfuric acid. Ethyl Alcohol. This is purified by adding 7 grams of silver nitrate and 15 grams of potassium hydroxide, separately (each dissolved in 100 ml. of ethyl alcohol), t o 4 liters of absolute ethyl alcohol. These are mixed, allowed t o stand overnight, and then distilled, using a T'igreux column. The first 700 ml. and the last 100 ml. are discarded. The effectiveness of purification is determined by reaction of this ethyl alcohol with the phenylhvdrazine-sulfuric acid. S o color should develop on standing overnight a t room temperature. Phenylhydrazine Hydrochloride. This reagent (Baker's, c.P.) is purified by adding 100 grams of phenylhydrazine hydrochloride t o a minimum amount of warm water (200 ml. at 60" t o 70" C.). With frequent stirring, it requires from 1 t o 3 hours to dissolve t h e phenylhydrazine. Do not heat. Heat 1 liter of ethyl alcohol t o boiling, and add t o t h e dissolved phenylhydrazine in water. Quickly filter while hot through Khatman S o . 2 filter paper. Cool in refrigerator, and collect the crystals on a sintered-glass filter (medium). Repeat the recrystallization procedure twice, dissolving the crystals in less water each time. The last collection of crystals should be n ashed with cold ethyl alcohol and thoroughly dried. Store the white crystals in a tightly stoppered brown bottle over calcium chloride. Proper purification
I
1
I
I
0 HYDROCORTISONE PLASMA
TIME REAGENTS
1
I
IN
HYDROCORTISONE
HOURS
Figure 1. Rate of color development of authentic hydrocortisone and plasma phenylhydrazine-reacting steroid with phenylhydrazine-sulfuric acid-ethyl alcohol reagent (25' C.)
Table 1.
Molar Absorbancy Indices of
Czl Steroids (410 Mp)
95O c., 10 hlin.
60" C., 20 ?*fin
37" c., 4 Hr.
26" c., 16 Hr.
7,500
19,400
22 , 100
22,500
(cortisone) Prennane-3a, 17a,21-triol-11,20-dione (cetrahydrocortisone)* Pregnane-Sa, 17a,21-tetrol-20-one
18,800
23,400
23,400
23,400
15 700
21, $00
22,000
22,200
(tetrahydrohydrocortisone) Pregnane-lia-o1-3,11,20-trione-21-
1,000
l1,iOO
14 400
15,400
acetate (dihydrocortisone) Pregnane-1 lp, 17a-diol-3,20-dione-21 acetate (dihydrohydrocortisone)
13,900
20 > 000
22,000
22 , 700
5,700
15,000
17,800
18,200
13 500
20,400
23,000
23,300
Steroida A4-Pregnene-llp,17a,21-triol-3,20-dione (hydrocortisone) ~4-Pregnene-17a,21-diol-3,11,20-trione
A4-Pregnene-l7a,21-diol-3,20-dione (compound S )
a 3 ml. of phenylhydrazine-sulfuric acid-ethyl alcohol added to 30 -, of dried steroid in test tube. Measurements made in Beckman DU spectrophotometer. iicetates of hydrocortisone and cortisone give same molar indices as the free steroid n hen corrected to the free compound. Absorption maximum at 415 mp.
must be evaluated by a melting point determination (240" to 243" C.). Blank Reagent. Two parts of 64y0 sulfuric acid with one p a r t of ethanol. This reagent keeps indefinitely. Phenylhydrazine-Sulfuric AcidEthyl Alcohol. Fifty milligrams of phenylhydrazine hydrochloride are dissolved in 50 ml. of t h e blank reagent. This reagent is prepared fresh daily. Hydrocortisone Standard. One hundred milligrams of hydrocortisone are dissolved in 100 ml. of absolute ethyl alcohol. -4 dilute !Torking standard is prepared by diluting 1 ml. t o 200 ml. with water ( 5 y per milliliter of water). PROCEDURE
All glassware must be scrupulously cleaned first with soap and water and again with concentrated sulfuric acid.
Extraction. Carefully add 5 ml. of heparinized plasma to 25 ml. of dichloromethane in a 200-ml. Erlenmeyer flask and place on a rotating table (Arthur H. Thomas Co. S o . 3623). Extract for 5 to 10 minutes with gentle rotation. Gently transfer entire contents to a 25ml. graduated cylinder. Avoid violent shaking, as this mag produce emulsions which are difficult to separate. Washing. After removing as much plasma as possible b y aspiration, add 2 ml. of 0 . l N sodium hydroxide t o t h e dichloromethane extract, shake vigorously for 15 t o 20 seconds, and then remove t h e alkali layer by aspiration. Aliquots. Transfer two 10-ml. aliquots (for unknown and blank) t o separate 15-ml. ground glass-stoppered conical test tubes. Color Development. F o r unknowns, add 0.2 ml. of phenylhydrazine-sulfuric acid-ethyl alcohol reagent t o dichloromethane extract; VOL. 29, NO. 1, JANUARY 1957
145
for blanks, add 0.2 ml. of blank reagent. Stopper the tubes, shake vigorously for 15 to 20 seconds, and allow t o stand for 30 minutes. Remove the supernatant dichloromethane phase by aspiration, and allow the sulfuric acid-ethyl alcohol solution t o stand a t room temperature for 8 to 24 hours for maximum color development. Spectrophotometry. Transfer the contents of the tubes t o microcuvettes (Pyrocell Manufacturing Co., New P o r k , 1.5 X 10 X 15 mm.), and measure the absorbance of the colored product against a water blank a t 410 mp in a Beckman DU spectrophotonieter. Five milliliters of water run through the entire procedure serves as reagent blank, and 5 ml. of water containing 5 Y of hydrocortisone serves as standard. Calculations
aqueous plasma and alkali layers, thus avoiding the use of separatory funnels. Dichloromethane also offers an advantage when the steroid is extracted with the sulfuric acid-ethyl alcohol reagent, because it has a density greater than that of the organic layer. There is no tendency to emulsion formation, and the top organic layer can be readily aspirated from the sulfuric acid-ethyl alcohol reagent. Rate of Reaction. Figure 1 shows the rate of color development of authentic hydrocortisone and the plasma phenylhydrazine-reacting steroid with the phenylhydrazine-sulfuric acid-ethyl alcohol reagent a t room temperature (25" (2.). Both the rate of development of color and the rate of fading of color (both show a loss of 8% of the maximal color a t 48 hours) a t 410 mp are essen-
Phen!-lh!-drazine-Treated Tubes, rl
0.2 ml. of phenylhydrazine
+ sulfuric acid + ethyl alcoho
Plasma sample A Reagent blank A Standard A Plasma sample -4- plasma sample B = a Reagent blank A - reagent blank B = b a - b = corrected absorbance (-4) of plasma sample Standard A - standard B = corrected absorbance ( A ) of standard c c - b = corrected absorbance ( A ) of standard A plasma X 5 y X 20 = micrograms of hydrocortisone per 100 ml. A standard EXPERIMENTAL
Choice of Solvent. Hydrocortisone has a distribution coefficient (expressed concentration upper phase as K = concentration lower phase with solvents saturated with each other a t 25" C.) of 0.14 in a system of water and dichloromethane. Chloroform (K = 0.13), ethylene dichloride ( K = 0.28), and ethyl acetate (K = 12) have very much the same efficiency for removing hydrocortisone from water. However, dichloromethane once adequately purified by treatment with silica gel remains stable for many months a t room temperature, whereas chloroform and ethylene dichloride will keep only a few days or weeks. Ethyl acetate is difficult to purify and cannot be purified by the very simple method of passing over silica gel. Also, it is miscible with the sulfuric acid-ethyl alcohol reagent. Benzene and ethyl ether have less favorable partition coefficients for hydrocortisone (benzene, K = 0.6; ethyl ether, K = 1.3). By using 5 volumes of dichloromethane with a single extraction, it is possible to extract 98% of the hydrocortisone from water into the organic phase. I n the extraction of the steroid from the plasma, it is desirable to use a solvent such as dichloromethane that is heavier than mater, as this makes it possible to aspirate and discard the top 146
ANALYTICAL CHEMISTRY
Blank Tubes, B 0.2 ml. of sulfuric acid ethyl alcohol Plasma sample B Reagent blank B Standard B
+
tially the same for plasma hydrocortisone and authentic hydrocortisone. Effect of Temperature. An increase in temperature shortens the time necessary for maximum color development. At 25" C. maximum color develops in 8 hours, a t 37" C. in 3.5 hours, and a t 60" C. in 10 minutes. The absorption maxima a t all three temperatures was the same-410 mp. The maximum color obtained a t 60" C., however, was less than that obtained a t 25" and 37" C. (Table I). Optimum Concentrations of Water, Ethyl Alcohol, and Sulfuric Acid. A concentration of sulfuric acid of about 42.5 =t 2y0 produced maximal color. Ethyl alcohol must, however, be present in a concentration of 30 to 50% for maximal color. It is desirable to use some water in the reagent because of the intense heat of reaction produced by mixing equal parts of concentrated sulfuric acid and ethyl alcohol. Optimum Concentration of Phenylhydrazine and Preparation of Hydrocortisone Bisphenylhydrazone. Maximal color development requires the presence of 20 y of phenylhydrazine per 1 y of hydrocortisone; higher concentrations of phenylhydrazine do not produce any additional color. The phenylhydrazine derivative of hydrocortisone r a s prepared under the conditions of the recommended assay. The derivative was precipitated from
the sulfuric acid-ethyl alcohol-water reagent by adding excess water. The precipitate was washed ITith water and dried in vacuo. Attempts to recrystallize this compound from various solvents were unsuccessful. Analysis of this compound (melting point 155" to 160" C.) revealed the following: C
H
S
Calculated
(CaHdT408) 73.3 7.46 10.36 Calculated (hydrate with 1 mole of water) 70.94 7.58 10 03 Found 70 5 7.46 9 87
Relationship of Ratio of Volume of Reagent to Volume of Dichloromethane. Hydrocortisone can be extracted from dichloromethane with the sulfuric acid-ethyl alcohol-water reagent over a wide range of ratios of volume of reagent to volume of dichloromethane (Table 11). When a constant volume of dichloromethane (10 ml.) is used and the volume of reagent (0.2 to 2.0 ml.) is varied, similar molar indices are also obtained. The presence of phenylhydrazine in the reagent is without effect on the extraction. Conformity to Beer's law was indicated by the linear relationship between the micrograms of hydrocortisone determined and the absorbance when the steroid was determined with or without extraction from dichloromethane. Sensitivity of Reaction. The molar absorbancy index of the bisphenylhydrazone of hydrocortisone was 22,500 (Table I), or 23.500 in the presence of traces of dichloroniethane (Table 11) (25" C. after 8 to 24 hours' incubation). This
Table II. Extraction of Hydrocortisone (2 7 ) from Various Volumes of Dichloromethane with Phenylhydrazine Reagent Ratio of Dichloro- Molar Dichloro methane ilbsorbExmethane, to PNH ency tracted, M1. Reagent" Index& % 0 1
2 5 10
20
30 40
5:l
1O:l
25:l 50:l 100:l 150:l 200:l
23,500~
23,500 23,100 22,600 22,400 22,200 21,900 21,500
100 100
98 3 96 3
95 3 94 5 93 3 91.5
a 0.2 ml. of phenylhydrazine-sulfuric acid-ethyl alcohol reagent (25" C., 16 hr.). * The higher molar indices (see Table I) are result of slight color potentiation of dichloromethane. c Determined with phenglhydrazinesulfuric acid-ethyl alcohol saturated with dichloromethane.
I - 7 - ,
-.-
4
p.9
HYDROCORTISONE
- 4 p 9 HYDROCORTISONE
BLANK
25ml.PLASMA
0.400
\
/
25ml.PLASMA BLANK
\
/
\
/
\
\
w 0.300
u z a
\
m LT
0 v)
m
a
0.200
c
350
\
I
I
375
400
I 425
WAVE
Figure 2. material
\
I 450
LENGTH
I
I
475
500
mp
Spectral absorption curves of phenylhydrazine-reacting
Spectra run in Beckman DK recording spectrophotometer
- - - Plasma extract ----. _ Authentic hydrocortisone
Plasma extract blank rnoterial Authentic hydrocortisone in sulfuric ocid-ethyl alcohol reagent
.9
I
L
I
----
.0
I
1
1
I
HYPERBILIRUBINEMIA NO HYDROCORTISONE AFTER ACTH POOLED NORMAL PLASMA (HEPARIN)
.7
.6 W
g .5
a
m
g .4 cn
m
a
.3
.2 .I
.o 350
370
390
410
430
450
470
490
510
530
WAVE L E N G T H m p Figure 3. Spectral absorption curves of plasma background (blank) material reacting with sulfuric acid-ethyl alcohol reagent Spectra were run in a Beckman DK recording spectrophotometer
corresponds to a sensitivity of about 0.04 p.p.m. This order of sensitivity and the use of a small volume (0.2 ml.) of reagent for the final colorimetric assay make it unnecessary to add hydrocortisone to the plasma sample before extraction, as was suggested by Silber and Porter (IO). Absorption Spectra of Hydrocortisone and Plasma Steroid. Figure 2 shows the absorption spectra of the bisphenylhydrazone of hydrocortisone and of the phenylhydrazine-reacting material in the dichloromethane extract of plasma. I n addition, the absorption spectra of hydrocortisone and the “background” (blank) substances in the dichloromethane extract of plasma, extracted by the sulfuric acid-ethyl alcohol reagent without added phenylhydrazine, are shown. There is a good correspondence between the spectral curves of authentic hydrocortisone and the background substances in the dichloromethane extract of pooled plasma from normal subjects. The curves are nearly linear from 360 to 460 mp. These observations would seem to validate the use of the Allen ( 1 ) correction factor for the blank material reacting with sulfuric acid, as proposed by Kelson and Samuels (6). However, plasma extracts from patients with hyperbilirubinemia, and after adrenocorticotropin administration, do not show this linear character (Figure 3). Thus, unless a spectral curve is run on each plasma, it is necessary t o introduce a plasma sulfuric acid-ethyl alcohol blank correction, Bayliss and Steinbeck (2) have also found it necessary to use a sulfuric acid-ethyl alcohol plasma blank rather than a simple correction factor. Specificity of Reaction. The yellow chromophore (bisphenylhydrazone) formed with phenylhydrazine and hydrocortisone is relatively specific, as only a fex other naturally occurring steroids give the characteristic color with an absorption maxima a t 410 m l ( 8 ) . Table I Lists the molar indices and absorption maxima of various naturally occurring steroids that give this reaction. Apparently these other steroids exist in very low concentration in plasma, are absent, or are not extracted with dichloromethane-viz., A4-pregnene-6B,1ID, 17a,21-tetrol-3,20-dione-a~ this method has been shown to be highly specific for hydrocortisone in plasma. This specificity has been established by a n isotope dilution technique in plasma from normal subjects and in patients with the follom-ingdiseases: myxedema, thyrotoxicosis, acute febrile states, jaundice due to liver disease, rheumatoid arthritis, and various anemias. A small quantity of hydrocortisone-4C“ (1.0 y, specific activity 4600 c.p.m. per 7 ) was added to 15 to 20 ml. of VOL. 29,
NO. 1, JANUARY 1 9 5 7
147
plasma, and the plasma was extracted with 5 volumes of dichloromethane. The dichloromethane, after being xashed successively with volume of 0.02N sodium hydroxide, volume of 0.01M acetic acid, and ljlS volume of water, was then chromatographed on paper at room temperature for 8 to 10 hours in a modified Bush-type (4) system (four benzene to two methanol to one water). The hydrocortisone of the extract, running a t the same rate as authentic hydrocortisone (approximately 15 cm. in 8 hours) was located by ultraviolet light scanning. This area was cut out and eluted with a small volume of cold 95% ethyl alcohol, and the eluate was evaporated t o dryness under nitrogen. The residue was dissolved in dichloromethane and the specific activity of the hydrocortisone determined from a radioactivity assay and phenylhydrazine assay of aliquots of the dichloromethane. From this specific activity value and a knowledge of the quantity and specific activity of the added tracer, the quantity of hydrocortisone in the plasma was calculated : H
H
=
h 6-
dilution assay is shonm in Table I11 (although in some cases-e.g., diabetic acidosis-such close correspondence has not always been obtained). The basis for the specificity of this isotope dilution procedure has been presented in a previous publication (T), With one normal plasma pool, aliquots of this eluted steroid were also suhjected to fluorescence assay (7, 11). to acetylation to form the acetate, and to enzymatic reduction (12) to form the tetrahydro derivative of hydrocortisone. These fractions were again chromatographed and the specific activity was determined. With these three additional methods, the hydrocortisone concentration was found to agree with the phenylhydrazine method n ithiri 12%. Plasma from a patient who had
previously received 2.0 mg. of A1,9afluorohydrocortisone for 3 days (Table 111) was subjected to the phenylhydrazine assay procedure, and no hydrocortisone could be found. When the same plasma was subjected to an isotope dilution procedure of analysis, no hydrocortisone was found. Recoveries and Precision. Figure 4 demonstrates the linear relationship between the hydrocortisone concentration and the volume of plasma assayed. Table IV lists the recoveries of hydrocortisone added to plasma. Dilution of plasma with equal or double the volume of Fvater prior to extraction with dichloromethane results in recovery of the same quantity of hydrocortisone. At a plasma hydrocortisone concena precision within tration of 5-& k5T0was obtained; a t a concentration
0.5
1)
= micrograms of hydrocortisone
in plasma
h = micrograms of hydrocortisone4-CI4 added to plasma
I'
= specific activity of added hydro-
I
= specific activity of hydrocorti-
cortisone-4-C'? sone eluted from paper The close correspondence between the plasma hydrocortisone values obtained with the recommended phenylhydrazine procedure and the isotope PLASMA EQUIVALENTS, M L . Figure 4. Relationship of absorbance to increasing amounts of plasma with constant volume of dichloromethane and phenylhydrazine-sulfuric acid-ethyl alcohol reagent
Table 111. Plasma Hydrocortisone as Determined by Phenylhydrazine and Isotope Dilution Methods
Subject
Normal Normal Normal Normal Normal Normal
18
6.0 18 14 14
Normal, after intravenous ACTH Adrenocortical tumor Adrenogenital syndrome Panhypopituitarism Normal, 3 days after administration of 2.0 mg. A1,9afluorohydrocortisone per day 148
Hydrocortisone, ?/lo0 Ml. Plasma Phenyl- Isotope hydrazine dilution
Table IV.
18 6.0
5 MI.
21 15
Plasma
11
13 9.5
45 63
43
0.082 0,085 0.087 0.085 0.085
4.0
1.5
60 3.5 0
0.084
Mean
Recovery of Hydrocortisone Added to Plasma, Expressed in Absorbance
5 r Hydrocortisone 0.490 0.497 0.492 0.501 0.495 0.495
5 M1. Plasma Found
Calcd.'
0.553 0.555 0,559 0.547
0,580
0.552 0.551
0.551 0.552 0.560 0.557
0
ANALYTICAL CHEMISTRY
0
+ 5 y Hydrocortisone Recovered, 0.580
0,580
0.580 0,580 0,580 0,580
0.580 0,580 0,580
Based on mean of plasma, and hydrocortisone absorbance.
% 95.4 95.7 96.5 94.4 95.3 95.1 95.1 95.3 96.6 96.0 Mean 95.5
of I5y%, 1 1 . 5 % was obtained; and a t a level of joy%, ~ ' ~ 0 . 5 % was obtained. Assays of plasma hydrocortisone on aliquots of plasma taken a t weekly intervals from a single pool (137y0) over a 6 n i o n t h period (25 determinations) showed a deviation of &7.&.
Normal Values.
In 50 normal male and female subjects. the plasma hydrocortisone levels have ianged from 6 to 2 5 ~ 7 with ~ . a inem of 15 i 4.57% (standaid deviation).
( 8 ) Porter. C. C.. Silber. R. H.. J . Biol.
LITERATURE CITED
Allen. IT. >I., J . Clin.Endocrinol. 10, i 1 (1950).
Bayliss, R. I. S., Steinbeck, A. IT., Biochem. J . (London) 54, 523 (1953). (3) Bliss, E. L., Sandberg, A. il., Xelson, D. H., Eik-Xes, K., J . Clin. Invest. 32, 818 (1953). Bush, I. E., Biochem. J . ( L o n d o n ) 50, 370 (1952). Bush, I. E., Sandberg, A , , J . Biol. Chem. 205, i 8 3 (1953). Selson. D. H.. Samuels. L. T.. J .
Cherk. 185,'201 (1950). ' (9) Romanoff. E. B.. Hudson. P.. Pincus. G., J.' Clin'. Endocrinol. and Xetabolism 13. 1546 (1953). (10) Silber, R. H., Porter, C'. C., J . Biol. Chem. 210, 923 (1954). (11) Sweat, 11.L , -1s.4~.CHEJI.26, 773
(1954).
(12) Tomkins, G., Isselbacher, K. J., J . Am. Chem. SOC.76,3100 (1954).
(13) Weicheelbaum, T. E , Margraf, H. W., J . Clin. Endocrinol. and Metabolism 15, 970 (1955). RECEIVED for revieiv March 10, 1956. hccepted August 29, 1956.
J . Clin.Invest. 35, 552 (
Determination of Antimony in Indium Antimonide M. C. BACHELDER and PATRICIA M. SPARROW Institute for the Study of Metals, University of Chicago, Chicago, 111.
b Indium antimonide may b e brought into soluble form by fusion with equal parts of anhydrous sodium carbonate and sulfur. The fusion product i s dissolved in a minimum amount of concentrated hydrochloric acid, and the precipitated sulfur is oxidized with potassium chlorate. Chlorine is removed by boiling the solution and the antimony i s determined b y the iodometric method. The accuracy of the method for approximately 200 mg. of antimony i s 0.2%.
T
HE usual methods for dissolving antimony and its alloys are not applicable to indium antimonide. Seither hot concentrated sulfuric acid nor concentrated hydrochloric acid containing bromine or potassium chlorate dissolves the compound. Although the indiuni antimonide is dissolved by aqua regia, some antimony is lost by either volatilization of the chloride or precipitation of insoluble oxides of antimony prior to the analysis. Xitric acid containing tartaric acid also gives solution: but t n o valence states of antimony are formed, and the nitrate ion must be removed before the final determination of antimony. This proccldure lead3 to low results. The most expedient approach to sample solution and analysis mas found to be fusion with sodium carbonate and sulfur. This gives a hydrochloric acidsoluble melt that can be analyzed for antimony by a n iodonietric titration without separating the indium.
ground indium antimonide with six times its rreight of a mixture of equal parts of anhydrous sodium carbonate and pure sulfur ( 2 ) . Fuse the mixture in a covered porcelain crucible; initially heat with a low flame and gradually increase the height of the flame to a teniperature where the mass is in quiet fusion. Finally heat with a full flame until the excess sulfur is completely burned away. About 2 hours are needed to reach this point. Cool, place the crucible and cover in a 200-ml. Berzelius beaker, and add 20 ml. of 1 2 s hydrochloiic acid. K h e n the evolution of the hydrogen sulfide has subsided. remove the crucible and lid and wash with a minimum aniount of 1 2 s hydrochloric acid. -4dd 300 mg. of solid potassium chloride, and heat on the steam bath for 15 minutes to complete the reaction and expel the hydrogen sulfide. To the warm solution add solid potassium chlorate in small quantities until the solution is clear. Expel the chlorine by boiling. Transfer the hot solution to a 200-ml. Berzelius beaker using a minimum amount of 12.1- hydrochloric acid for rinse, and leave the small lump of agglomerated sulfur behind. 4 d d suffi-
Table 1.
EXPERIMENTAL DETAILS
During the fusion and while the crucible is m-arni, the cover should not be removed. Contact with air is sufficient to oxidize small amounts of the antimony to the quinquevalent form, which is difficult to dissolve. It is convenient to follox the fusion process by using a transparent quartz cover for the crucible. Dilute hydrochloiic acid or lvater must not be added to the products of the fusion, as antimony oxides formed by the hydjolysis Iyill precipitate. Solid potassium chloride is added to form the less volatile complex SbC4-, so that the hydrochloric acid solution may be heated on the steam bath without fear of losing antimony ( 8 ) . To remove the colloidal sulfur formed by the reaction of the sulfides with hydrochloiic acid and to ensure coniplete oxidation of the antimony to the quinquevalent state, solid potassium
Results of Antimony Determination"
Color End Point KO.of
cient 1 2 s hydrochloric acid to bring the total volume to 65 nil. Insert a thernionieter and bring this solution to constant boiling. Determine the antimony by the iodonietric method ( I ) .
Sb.
Potentiometric Titration s o . of
Sh.
mean % samples mein' % Sh 5 99.84i0.30 3 99.93 =k 0.03 Sb In20a 3 99.85 i 0 . 0 4 3 100.12 & 0.04 Sb InSb* 5 5 0 . 9 1 i 0.17 4 51.46 + 0 . 2 4 InSb 5 50.99 & 0 . 1 7 4 51.44 i 0.16 Approximately 200 mg. of antimony. i, % Sb in InSb = (total weight Sb found) - (weight Sb added) x 100. (weight of InSb) Sample
samples
++
5
PROCEDURE
Intimately mix 400 mg. of finely VOL. 29, NO. 1 , JANUARY 1957
149
