HYDROCORTISONE

Klaus Florey

1. Introduction 278
1.1 Foreword 278
1.2 Histoory 278
2. Description 278
2.1 Name, Formula, Molecular Weight 278
2.2 Appearance, Color, Odor 279
3. Isolation and Synthesis 279
3.1 Isolation 279
3.2 Chemical Synthesis 279
3.3 Biosynthesis 280
4. Physical Properties 28 I
4.1 Spectra 28 1
4.2 Solid Properties 289
4.3 Solution Properties 293
5. Methods of Analysis 294
5.1 Historical Synopsis 294
5.2 Elemental Analysis 296
5.3 Ultraviolet 296
5.4 Colorimetric 297
5.5 Fluorescence 299
5.6 Polarographic 300
5.7 Isotope Dilution 300
5.8 Chromatographic Methods 300
5.9 Electrophoretic 306
5.10 Bioassay-Enzymatic 308
5. I 1 Saturation Analysis 308
6. Stability-Degradation 309
7. Metabolic Products-Pharmacokinetics 310
8. Determination in Biological Fluids and Tissues 313
9. Determination in Pharmaceutical Formulation 3 14
References 315

ANALYTICAL PROFILES OF DRUG SUBSTANCES 277 Copyright by the American PharmaccuticalAssociation.

ISBN 0-12-260812-7
VOLUME 12
278 KLAUS FLOREY

1. Introduction
1.1Foreword
The compilation of an analytical profile
for hydrocortisone, also known as cortisol, turned
out to be a more ambitious undertaking than I had
expected. When scanning through Chemical Abstracts,
the literature is simply staggering. Fortunately,
there are quite a few very good and easily acces-
sible books and reviews available which will lead
the reader to secondary sources. First and fore-
most, there is the classic book on steroids by
Fieser and Fieser.' Helpful are also the more
modern books,on steroid analysis by Heftman* and
Gordg and Szaz.3 As in the case of aspirin, I have
endeavored to cover the newer literature as compre-
hensively as possible and have included only those
older references which are of historical interest.
Again, my apologies to all those scientists whose
publications in this field are not included here.
1.2History
Althouqh hydrocortisone is the major
hormone secreted by-the human adrenal cortex , it
was by no means the first adrenocortical hormone to
be isolated by the early investigators.
Reichstein14 the first to publish its isolation
from adrenal glands, gave it the letter M (see also
Section 3 ) . When Kendall and Hench made their
momentous discovery of the relief of the symptom of
rheumatoid arthritis5 in 1948, cortisone was the
first hormone to be made available by Merck and
Company. Only in the early fifties was hydrocorti-
sone also introduced into therapy, but was soon
eclipsed in potency and effectiveness by analogs
such as the A 1 - and 9a-halo compounds. Yet, it
still is much prescribed, since it is relatively
inexpensive. It is still the ultimate standard by
which the efficacy of other corticosteroids is
measured.
2. Description
2.1 Name, Formula, Molecular Weight
Hydrocortisone is pregn-4-3,20 dione,
11,17,21-trihydroxy-, ( 1 1 B l - i also 4-pregnene-118,
1701, 21-triol-3,20-dioneI 17-hydroxycorticosterone;
Reichstein's compound M; Kendall's compound F;
HYDROCORTISONE 219

cortisol Chem. Abstracts Registry Number [50-23-71.

21 CH20H
I
c=o

‘21H3005 M.W. 362.46

2.2 Appearance, Color, Odor, Taste

Bitter tasting, white to practically
white, odorless, crystalline powder.
3. Isolation and Synthesis
3.1 Isolation
In the late thirties, the isolation of
adrenocortical hormones was actively persued by
Pfiffner, Vars and Wintersteiner at Columbia, Mason,
Myers and Kendall at the Mayo Foundation, Cortland
and Kuizenga at the Upjohn Co. and Reichstein at
the University of Basle, Switzerland. It was
Reichstein4 who first described the isolation of
hydrocortisone and elucidated its structure. He
called it Substance M; but for several years, it
was better known as Kendall’s F. For details
relating to structure elucidation, I refer to
Fieser and Fieserl and also to Reichstein and
Shoppee.
3.2 Chemical Synthesis
As already mentioned (Section 1,2) ,
cortisone was the first corticosteroid introduced
into therapy, since the 11-keto group is more
readily accessible to synthesis. Hydrocortisone
was first synthesized in the Merck laboratories by
Wendler, Graber, Jones and Tishler7 from 20-cyano-
A17-pregnene-21-ol-3,11 dione, which in turn had
been prepared by Sarett8 in connection with his
cortisone synthesis. Introduction of oxygen at the
11 position has been crucial for the commercial
280 KLAUS FLOREY

production of corticosteroids from abundant natural

sources. The microbiological hydroxylation of
steroids at position 11, therefore, has been a
major breakthrough, pioneered in the Upjohn, Squibb
and Pfizer laboratories (for details, see
references 9 and 10) .The Pharmaceutical Manufac-
turinq Encyclopedia lists the microbiological
118 hvdroxvlation of Reichstein's compound S (11-
desox;- 17-hydroxy-corticosterone) to hydrocortisone
as a mode of manufacturing.

CH2OH CH2OH
I I
c=o C=O

However, I am not aware that this route is really

used on an industrial scale. The main commercial
routes to hydrocortisone are either based on
naturally occuring steroids, having an oxygen
function at carbon 12, or on llu-hydroxylation of
a suitable intermediate. In the first category are
the Merck process using bile acids and the process
starting with hecogenin, a constituent of sisal.
In the second category is the Upjohn process
starting with progesterone, derived from diosgenin,
a constituent of dioscorea or stigmasterol derived
from soybeans. Syntex played a leading role in the
development of suitable intermediates for the
diosgenin and hecogenin derived processes.
3.3Biosynthesis
The mammalian biosynthesis of hydrocortisone
and the other corticosteroids occurs in the adrenal
gland. The common precursor is cholesterol. The
immediate precursors of hydrocortisone are primarily
ll-desoxy-cortisol (Reichstein's compound S),which
is 118 hydroxylated enzymatically, and cortico-
sterone, which is hydroxylated at carbon 17.12 It
is curious to note that no mammalian lla-hydroxy-
lating enzymes are known which are the prevalent
HYDROCORTISONE 281

enzymes in microorganisms (see 3.2). The elucida-

tion of biosynthetic pathways was pioneered by
Hechter and Pincus and their collaborators at the
Worcester Foundation in the late forties.13 For
details on the biosynthesis of adrenal steroids,
I refer to an excellent review by Samuels and
Uchikawa14 and the book on steroid biochemistry by
Heftmann.15
4. Physical Properties
4.1 Spectra
4.11 Infared
The first spectrum of hydrocortisone,
prepared in a nujol mull was published by Collings-
worth, et a1 in 1953.16 A sDectrum of hvdrocorti-
.
sone dispersed in a potassium bromide disk was
recorded by Hayden17 in 1954. The classical atlas
of infrared spectra of steroids by Dobriner,
Katzenellenbogen and Jones18 contains only the
spectrum of hydrocortisone acetate. Mesleyl’
described infrared spectra of hydrocortisone
acetone, had no absorption near 760 cm-B.
polymorphs. Form A, evaporated from eth no1 or
Form B,
evaporated from chloroform, gave a double peak at
761 and 752 due to chloroform.
The spectrum of the USP reference
standard of hydrocortisone in a mineral oil
dispersion,218presented in Figure 1, essentially
agrees with the spectra in reference 16.
4.12 Ultraviolet Spectrum
Hydrocortisone exhibits the ultra-
violet absorption spectrum typical of a,B-unsatu-
rated ketones. Such a spectrum, with a maximum at
241 nm, was recorded already in 1936 for Reichstein4
by Mehler of Zurich, Switzerland on a Hilger quartz
spectrograph. The Merck Index gives a X maximum
242 nm Ei 445.20 The chapter on steroids by
A.A. Forist and J.L. Johnson in Pharmaceutical
Analysis2I gives a X maximum 242 nm, E maximum
16,200. See a l s o the review article on ultra-
violet absorption of steroids by L. Dorfman.22 A
A maximum 242 nm, E maximum 15,800 has also been
reported.23 It also has a weak maximum at 290 nm
(log E 2.O1Iz4 and inflections at:
 WAVELENGTH (MICRONS)

0.2

0.4

0.6
0.8
1.o

Figure 1. Infrared spectrum of hydrocortisone (USP reference standard) in a

mineral oil dispersion (Instrument: Perkin-Elmer 621).
HYDROCORTISONE 283

x (nm)
- log E
315 1.82
324 1.75
332 1.73
338 1.65
346 1.56
357 1.23
4.13Fluorescence
Hydrocortisone does not have any
native fluorescence, but it can be induced by
reaction with concentrated acids (see Section 5.5).
4.14Phosphorescence
The singlet + triplet transitions by
phosphogescence excitation spectroscopy at both 77

1
and 4.2 K were studied for several steroids.25
The following data pertain to hydrocortisone:
Phosphorescence emission: 3880 origin; 4350 8
maximum; So+T absorption: 3826 ; S-tS (rl,II) dire t
absorption: 3,600, 3,440, 3,310 ; A E: 1600 cm E .
4.15 Nuclear Magnetic Resonance26
The 100 MHz proton NMR spectrum of
hydrocortisone in DMs0-d~is shown in Figure 2.
The spectrum contains characteristic resonances at
6 0.76 s (l8-CH3), 61.37 s (19-CH3), 65.55 s (C4-H),
64.26 M ((211-H) and a coupled A B quartet at 64.50,
4.05 (21-CH2). Three peaks that exchange with D20
were detected at 65.14, 4.64 and 4.26 (11, 17 and
21-OH). All proton chemical shifts are referenced
to internal TMS at 0.0 PPM.
The carbon-13 NMR spectrum of hydro-
cortisone (Figure 3) is summarized in Table 1.
Assignments of all 21 carbons are listed and are
consistent with those of Blunt and Stothers.27 The
reference peak in the carbon spectrum was the DMSO
multiplet which was assigned as 39.5 PPM from TMS.
The spectra shown in Figures 2 and 3
were obtained on a Varian XL-100-15 NMR spectro-
meter equipped with a Nicolet TT-100 data system.
4.16Mass28
The low-resolution mass spectrum of
hydrocortisone (see Figure 4) shows the expected M+
at m/z 362. Corticosteroids generally show frag-
Figure 2. Proton NMR spectrum of hydrocortisone. Instrument: Varian XL-100-15.
N
oo
01

Fig. 3. Carbon-13 NMR spectrum of hydrocortisone. Instrument: Varian XL-100-15

 6394 SQ9318 B R T C H *NX001 200 DEG

01-MRY-80 MB0764
1001

90-

80.-
>
I-

II)
70.-
Z
I
z
U

W
>
U

l-
a
J
w
CY

MRSS/CHRRGE
I N T E N S I T Y SUM = 4 0 8 2 5 BRSE PERK Z = 3 . 4 3

Figure 4. Low Resolution Mass Spectrum of Hydrocortisone. Instrument: MS-9

HYDROCORTISONE 287

TABLE 1. CARBON-13 NMR DATA FOR HYDROCORTISONE

CARBON # CHEMICAL SHIFT ( 6 )
1 34.0
2 33.5
3 198.0
4 121.5
5 172.3
6 32.8
7 31.4*
8 31.2"
9 55.6
10 38.9
11 66.5
12 39.1
13 46.3
14 51.6
15 23.3
16 33.0
17 88.5
18 16.9
19 20.4
20 211.5
21 65.8
6 in PPM from TMS (ext): Reference peak DMSO at
39.5 PPM.
* These assignments may be interchanged.

mentation patterns resulting from the l o s s of

D-ring substituents combined with the loss of small
neutral molecules such as water. The principle
hicrh mass ions are thus formed from the loss of
these groups, as depicted below. *
a

m/z 313
M+ m/z 362

m/z 302 m/z 344 +H

m/z 329
\r m/z 267
* Scheme is intended to show losses rather than to
explicitly show fragmentation pathways.
** Rearrangement
288 KLAUS FLOREY

Ions of mass less than m/z 267 (CigH230) occur

throucrh the loss of carbons (and attached hydrogens)
progressing from the D-ring. .

L m/z 267 -

In contrast to 1,4-dien-3-ones, the 4-en-3-ones

yield less intense diagnostic A-ring ions of m/z123
and 124. The analogous 1,4-dien-3-one would have
had a much more intense A-ring m/z 121, 122 ions.
The mass spectrum and fragmentation
pattern presented here is in essential agreement
with data reported earlier.29
A comparison of field desorption,
chemical ionization and electron impact mass spectra
of some steroids30 revealed that, both in the field
desorption and chemical ionization mode, the
molecular ions ( (M)+ and (M+H)+, respectively) are
the base peaks for hydrocortisone, while m/e 302 is
the base peak in the electron impact mode.
The field desorption kinetics in the
mass spectrum of hydrocortisone indicate that the
(M-C2H302)+ ions are formed by the gas phase
decomposition of the hydrocortisone molecular ion
at times >10-9-8 s.31
HYDROCORTISONE 289

4.2 Solid Properties

4.21 Melting Range
The following melting ranges have
been reported:
OC . Reference
217-220 with some decomposition 20
from ethanol and isopropanol
212-213 (commercial samples) 20
About 214 with decomposition 32,33
207- 214 34
215-218 24
201 (from ethanol, micro rn.p.1 35
4.22Differential Thermal Analysis
Hydrocortisone (USP reference
standard) when heated at 15O/min. (Instrument:
Dupont 900) gave a single endotherm at 221.50.36
4.23Thermogravimetric Analysis
Hydrocortisone (USP reference
standard) when heated at 15O/min. (Instrument:
Dupont 900) had a 0% weight l o s s to a temperature
of 222O. 36
4.24Calorimetry
The heat of combustion of hydro-
cortisone and other steroids was determined in a
microcalorimeter.37
4.25Crystal Properties
Microscopic pictures of hydro-
cortisone crystals were already presented by
Reichstein4 in 1936. As most steroids, hydro-
cortisone crystallizes in polymorphic and solvated
modifications. These can be summarized as follows:
 Solvent of
Modification Crystallization I R Characteristics

I Ethano1,acetone OH C=O and C=C Other :

3430 1715,1708,1645 902 (strong band) 19,38
3310 (sh) 1631,1611 887 (sh)

I1 Desolvation from 3435

chloroform 3350(sh) 1712,1643, 38
1630,1611

I11 Methanol Solvate Met hano1 3515 1722,1643 38

3405 1628,1607
3225

IV Chloroform Solvate Chlorofo m 3435 1712,1643 761 ,752 19,38,39

3350(sh) 1630,1609

The optical crystallographic properties of hydrocortisone crystal-

lized from ethanol were determined as follows:35
Crystal System and Habit; monoclinic plates;
Optic sign: +; Optic axial angle: 56O (58O calc.)
Dispersion: R>V; Refractive Indices: Nx: 1.540; Ny: 1.562; Nz: 1.638
Solvents, other than ethanol, gave hexagonal (dichloromethane) and
orthorhombic (methanol) crystals.42
Hydrocortisone was subjected to single crystal x-ray analysis and
HYDROCORTISONE 291

the established structure compared to that of 9a-

fluorohydrocortisone and other related steroids.41
Hydrocortisone methanol solvate (Cambridge Crystal
Library Code: (ORTPIS), when subjected to single
crystal x-ray analysis, was found to crystallize in
the orthorhombic space group P212121 with a=14.372
(4); b=18.400(5); c=7.706(2); 8,2=4. Ring A is a
distorted "sofa", rings B and C are in the "chair"
configuration, and ring D is close to a C(13)
envelope, with a pseudorotational parameter A of
26.1O. The ring junctions B/C and C/D are both
trans. The molecule as a whole is slighgly convex
towards the 8-side, with an angle of 10.9 between
the C(lO)-C(19) and C(13)-C(18) vectors.42 These
findings have been compared with energy calculations
according to the Westheimer The electronic
structures of hydrocortisone and other related
steroids was examined by extended Hueckel molecular
orbital calculations. Total and orbital energies,
Mulliken population, analytical data and dipole
moments were reported.44 The crystal structure of
the pyridine solvate (Cambridge Crystal Library
Code: CORTPY) has also been determined.45
The x-ray powder diffraction data
for hydrocortisone are presented in Table 2 and
Figure 5.46 An early x-ray powder attern was
presented by Collingsworth, et al. 1g Low micron
particles of hydrocortisone were obtained by ultra-
sonic irradiation of supersaturated solutions.47
TABLE 2

Powder X-Ray Diffraction

Pattern of Hydrocortisone (USP Reference Standard)
20 d (8) I 1/10
5.99 14.72 4.5 0.52
9.40 9.40 0.5 0.006
10.60 8.34 0.5 0.006
11.90 7.34 2.0 0.023
12.89 6.86 1.0 0.012
14.70 6.02 86.0 1.000
15.56 5.69 1.0 0.012
16.37 5.41 8.0 0.093
17.20 5.15 4.0 0.047
17.69 5.01 22.0 0.256
19.07 4.65 4.3 0.050
Figure 5. Powder X-Ray Diffraction Pattern of Hydrocortisone (USP Reference
Standard). Instrument: Norelco
HYDROCORTISONE 293

20 d (8) -
I 1/10
19. 80 4.48 4.5 0.052
20.49 4.33 2.0 0.023
20.98 4.23 1.0 0.012
23.52 3.78 2.5 0.029
24.50 3.63 0.5 0. 006
27.59 3.23 1.5 0.017

4.3 Solution Properties

4.31 Solubilitv
According to Clarke,48 hydrocortisone
has the following solubilities:
%

Water 0.029 (1 in 3 , 5 0 0 )
Ethano1 2.5 (1 in 4 0 )
Acetone 1.25 (1 in 8 0 )
Propylene glycol 1.0 (1 in 100)
Slightly soluble in chloroform, almost
insoluble in ether.
The solubility/tem erature profile
in ethanol ranges from 6.3% at 65' to 1 . 5 % at 25°.49
The solubility in water is increased by the addition
of surfactants such as Tween 20 ( 0 . 0 2 7 mol. of
steroid/mol. of Tween 20) .50 The solubility in
tris-buffered KC1 was found to be 161.0 1-1 molesfi
and,in egg lecithin liquid crystals, 1 5 . 5 n moles/
P mol. lipid.51 The solubilization of hydro-
cortisone and other steroids by long-chain polyoxy-
ethylene surfactantsS2 as well as in polyethylene-
glycol fatty acid esters53 has been studied.
Calculations on the dissolution of
hydrocortisone have been presented in connection
with comparing the measurement of the interfacial
energy between solid particles and a dissolving
solvent to the film theory of dissolution in which
diffusion is the principal mechanism.54 A non-sink
dissolution rate equation has also been applied to
hydrocortisone.55
4.32 Partition Coefficients, Diffusion
An extensive list of partition co-
294 KLAUS FLOREY

efficients (Table 3) were presented by Engel and

Carter. 5
For partition coefficients in
surfactants and solubilizers, see references 52
and 53 (Section 4.31). An estimation of hydro-
cortisone partition coefficients in ether:water
based upon molecular constitution has been
attempted.57 The absorption kinetics of hydro-
cortisone at the water-neutral oil interface has
been studiedI5*using the Gibbs equation59 and
verifying the Szyskowski law.60 The effect of
surfactants on the diffusion of hydrocortisone
across a cellulose acetate membrane has been
investigated.
Optical Rotation
4.33
The following values have been
reported:
Reference
( a )Do temp.
+167 (abs. ethanol) 22 20
+16 3O (methanol) 22 20
+150-15 6O (dioxane) 20 ,25 32,33 ,48
+162 24
Optical rotatory dispersion studies
were conducted by Foltz, Lippman and D J e r a ~ s i . ~ ~
The following values were obtained:
(a)700 + 113O; (a)589 + 162O; (a)300 +
12020; "max" (a)385 + 4560; "min" (a)367,5 +
312O; Ilmaxll(a1357.5 + 460°; 'lmin" (a!352,5 +
428O; "max" (a)315 + 3506O; c = 0.10 in dioxane;
temp. 23-25. Drude equation: (MI = 172/A2-
0.0551; A, 235 nm; % deviation (M) Obsd -
(M) calcd: -+ 1.0%; 620-410 nm; -+ 3.7%, 700-400
nm.
5. Methods of Analysis
5.1Historical Synopsis
As with all hormones which are present in
biological systems only in minute quantities, an
assay system, usually a biological one, is needed
as a guide for their detection and isolation. In
the early work leading to isolation of hydro-
cortisone and other corticosteroids from adrenal
HYDROCORTISONE 295

TABLE 3
Partition Coefficients for Hydrocortisone
(from reference 56)

1. Benzene/water 0.36
2. Benzene/50% methanol in water 0.14
3. Benzene/50% methanol in water 0.31
4. Petroleum ether/35% ethanol in water <o. 02
5. 25% Sec. butanol in n-hexane/water 0.24
6. 35% Sec. butanol in n-hexane/water 0.84
7. 25% Ethyl acetate in n-hexane/water 0.04
8. 50% Ethyl acetate in n-hexane/water 1.00
9. 30% Ethyl acetate in cyclohexane/30% ethanol
in water 0.31
10. 30% Ethyl acetate in cyclohexane/50% ethanol
in water 0.08
11. 30% Ethyl acetate in cyclohexane/70% ethanol
in water 0.04
12. 50% Ethyl acetate in cyclohexane/30% ethanol
in water 0.96
13. 50% Ethyl acetate in cyclohexane/50% ethanol
in water 0.39
14. 50% Ethyl acetate in cyclohexane/70% ethanol
in water 0.16
15. 70% Ethyl acetate in cyclohexane/30% ethanol
in water 2.7
16. 70% Ethyl acetate in cyclohexane/50% ethanol
in water 1.2
17. 70% Ethyl acetate in cyclohexane/70% ethanol
in water 0.02
18. Ethyl acetate/water 12.2
19. Water/carbon tetrachloride 0.16
20. 30% Methanol in water/70% chloroform in carbon
tetrachloride 0.70
21. 50% Methanol in water/50% chloroform in carbon
tetrachloride 2.6
22. 70% Methanol in water/30% chloroform in carbon
tetrachloride 5.9
23. 30% Ethanol in water/carbon tetrachloride 0.06
24. 0.05% NaCl in water/50% n-hexane in chloroform 2.57
25. 20% Ethanol in water/50% n-hexane in chloroform 2.57
26. 30% Ethanol in water/chloroform 0.06
27. 30% Ethanol in water/50% n-hexane in chloroform 1.63
28. 50% Ethanol in water/50% n-hexane in chloroform 0.61
29. 0.1N Acetate buffer/methylene chloride 0.12
30. 20% Methanol in water/50% n-hexane in chloroform 4.0
31. 80% Methanol in water/l.2-dichlvroethane 1.26
2% KLAUS FLOREY

tissue, the response of adrenalectomized animals

was used. Later on, corticosteroids were classi-
fied according to their relative activity in
promoting deposition of glycogen in the liver and
in controlling electrolyte balance in body fluids.
Hydrocortisone was found to have the highest gluco-
corticoid activitv and to be rather inactive
in controlling the electrolyte balance (cf. ref. 1,
p. 604).
When cortisone and hydrocortisone found
therapeutic applications, the Porter-Silber method62
permitted insight in the concentration of steroids
with the dihydroxyacetone side chain in biological
fluids. The blue tetrazolium method of blader and
provided a convenient and stability indica-
ting control procedure of these steroids in pharma-
ceutical preparations. Z a f f a r ~ n iapplied
~~ the
well-known color reaction of steroids with sulfuric
acid to hydrocortisone.
Paper chromatography, as an analytical
tool for steroids, was introduced in the fifties by
Zaffaroni and Bush, TLC and GLC followed in the
sixties, and HPLC in the seventies. For background
and discussion of methods, three excellent recent
books on steroid analysis are available. 2 ~ 3 ~ 6An
5
older book on pharmaceutical analysis also includes
a chapter on steroid analysis.21
5.2 Elemental Analysis
The elemental composition is as follows:20
C:69.59%; H:8.34%; 0:22.07%

5.3 Ultraviolet
The stronq abscmption band in the ultra-
violet ( A max 242 nm; Ei 445, see section 4.12) has
been used for quantitation of hydrocortisone itself
(cf. 21), but due to the interference of excipients,
it rarely can be used in the quantitative determi-
nation of dosage forms without difficulties. The
sodium borohydride method of GorZjg66 overcomes this
obstacle by using the reduced solution as a blank
in the reference cell. Kir~chbaurn~~ found that
addition of propylene glycol assures complete
reduction.
HYDROCORTISONE 297

5.4 Colorimetric
5.41 Sulfuric Acid
It was already known in the thirties
that steroids give color reactions with concentrated
sulfuric acid (cf. 3, p. 1 8 2 ) . A s already mentioned,
Z a f f a r ~ n iwas
~ ~ the first to apply it to hydro-
cortisone. He found absorption maxima at 2 8 0 , 3 9 5
and 4 7 5 mp. My personal prejudice concerning this
method coincides with that of Gorog and S z z z , who
....
state (cf. 3, p. 1 8 2 ) : I' spectra are not
reproducible as well as in the common solvents: the
positions and intensities of the maxima depend on
the time elapsed between dissolution of the steroid
and the recording of the spectrum, the temperature,
the quality of the sulfuric acid, the purity of the
sample, etc." The method today seems to be only of
historical interest, and I refer the reader to the
classical paper by Bernstein and Lenhard6* and the
chapter by Smith and Bernstein.69
They present the following data for
hydrocortisone:
1%
max (nm) Elcm
237 420
2 82 462
391 325
475 1 53

Sulfuric acid has also been used

diluted with other solvents, but rarely water. A
mixture of glacial acetic acid and concentrated
sulfuric acid ( 4 : 6 ) was found to give an absorption
maximum at 4 7 0 nm with hydrocortisone but not
cortisone.7 0
When hydrocortisone was heated in
sulfuric acid and treated with phosphorus
pentoxide, it gave an absorption maximum at 5 4 8 nm
(optical density 0 . 6 2 5 ) . 7 1
5.42 Other Acids
Hydrocortisone in orthophosphoric
acid (cf. 3, p. 1 8 6 ) gives spectra very similar to
sulfuric acid. The U.V. absorption maximum of
hydrocortisone in 7 2 % perchloric acid has been
given as 2 8 3 nm.72 Concentrated hydrochloric acid
has great specificity for the 116-hydroxyl group.
298 KLAUS FLOREY

Hydrocortisone gives a sharp, intense maximum at

465 nm in contrast to lla-h droxy, 11-keto, or
11-unsubstituted steroids.7 3

5.43 Porter-Silber
The principle of the Porter-Silber
method62 is the reaction of the dihydroxyacetone
side chain with phenylhydrazine in methanolic
sulfuric acid to produce a stable yellow color with
a maximum at 410 nm. This reaction is specific for
17-hydroxy steroids, and the sensitivity of the
method permitted its use to determine glucocorti-
costeroids in biological fluids. It was first
applied in 1952 to determine h y d r o c ~ r t i s o n ein
~~
urine and in 1957 to plasma75. With the advent of
chromatographic and RIA techniques, the method
may no longer have the importance it once had, but
it still is described in a modern (1975) book on
the determination of hormones.76
5.44Tetrazolium
The tetrazolium methods still belonq-
to the most important analytical techniques to
determine corticosteroids as such and in pharma-
ceutical preparations. For an excellent discussion
of mechanism and history, see reference 3 , p. 331.
It was first used for hydrocortisone as a spray
reagent for paper chromatography by Burton,
Zaffaroni and Keutman.77 Mader and Buck are
credited with extending the use of tetrazolium to
pharmaceutical steroid analysis but did not
describe the application to hydrocortisone in
their original paper.63 Hydrocortisone was first
quantitated by Henley,78 using 2-iodophenyl-3-
nitrophenyl-phenyl tetrazolium chloride. The USP33
favors the use of blue tetrazolium (B.T.; diani-
sole-bis-diphenyl tetrazolium chloride), the B.P.32
the use of triphenyl tetrazolium chloride (TZ) for
the determination of hydrocortisone and its dosage
forms. Absorbance maxima for the two variants are
at 525 nm and at 485 nm, respectively. The tetra-
zolium method is stability indicating, albeit an
indirect one measuring the reducing capacity of
the steroid side chain to form formazans. The
blue tetrazolium method has also been automated
for the assay of hydrocortisone in tablet^.^^^,^^
HYDROCORTISONE 299

5.45 Miscellaneous
For the determination of the free
21-hydroxy group of hydrocortisone in the presence
of its acetate,oxidation of the side chain with
cupric acetate to the glyoxal and condensation
with 0-phenylenediamine, (A max 331 nm, E 10,200) or
its 4,5-dimethyl derivative ( A max 351 nm, E 12,700)
have been used.80 Condensation with phenylhydrazine
gives an absorption maximum at 364 nm ( E = 19,000)
which has been used for the determination of hydro-
cortisone in tablets.81 For the determination of
residual hydrocortisone in prednisolone, the rate of
thiosemicarbazone formation was determined at
3 0 2 nm.82 A l s o used were reactions with Dische 83
reagent (diphenylamine in acetic and sulfuric acid),
bismuth oxidation,84 condensation with isonicotinic
acid hydrazide (370 nmIa5 also automated for
tabletsa6 and reaction with ammonium molybdate
(A max 655 nm) . 8 7
5.5 Fluorescence
Although hydrocortisone has no native
fluorescence, it can be induced by treatment with
concentrated sulfuric acid. This was already
observed by Wintersteiner and Pfiffners8 in 1936.
The various conditions of acid concentration and
temperature leading to slight variations of the
excitation wavelength (470 nm) and absorption
maximum (530 nm) as well as intensity of fluores-
cence are described by Goldzieher.89 The great
sensitivity of the reaction made it an ideal tool
to study the concentration of hydrocortisone in
biological fluids and tissues in a quantitative
fashion. It was first explored by Sweatgo in 1954.
He was able to determine as little as 5 nanograms
of hydrocortisone in 1 ml of ethanolic sulfuric
acid. The early work on the fluorimetric analysis
in several laboratories has been reviewed by
Silber.gl Phosphoric acid has also been used to
induce fluorescence,92r93as have been acetic acid-
antimony t r i ~ h l o r i d e ,aluminum
~~ salt and isonico-
tinylhydrazine (Ex.: 380 nm; Em: 495 nrnlg4 and
lithium hydroxide pellets (Ex.: 396/Em: 510) .95
A fluorescing derivative, amenable to TLC,96 has
been obtained by reactions with 4(6-methylbenzo-
thiazol-2yl) phenyl isocyanate.
300 KLAUS FLOREY

5.6Polarographic
Rased on the reduction of the 3-carbonyl
-
function, the polarographic reduction of hydro-
cortisone has been studied in well-buffered 50%
ethanol solutions.97 The halfwave potential ( E % )
was found to be dependent on pH, ranging from -1.26
for pH 2.8 to -1.74 for pH 10.8. A halfwave
potential of -1.50 was found in 90% ethanol at
pH 8.5.98 In DMF buffered at pH 9.15, a halfwave
potential of -1.64V was found.99 In acetonitrile-
tetrabutylammonium perchlorate, a halfwave potential
of -1.58 was found. The approximate detection limit
was determined as 3~10'~moles/liter . Polar-
ography of hydrocortisone has also been performed
with prior derivatization with Girard's reagent T
exhibiting a halfwave potential of 1.12lo1 and
betainyl hydrazine (E$ -1.42).98 Polarography has
been used for the determination of hydrocortisone
in ointments,99r102human bloodlol (see sections 8
and 9), and as a microbial conversion product after
TLC separations.lo3
5.7Isotope Dilution
Preparation of tritiated hydrocortisone
and its detection in fermentation brothlo4 and
adrenal sliceslo5 has been described.
5.8Chromatographic Methods
5.81 Paper
With the advent of TLC and HPLC, the
importance of paper chromatographic methods to
determine hydrocortisone declined steeply. It now
is only of historical interest. Yet, in the fifties
it was successfully used to enlarge the knowledge of
the distribution of hydrocortisone in body fluids
and tissues and to assay its purity, as well as
stability in dosage forms.
Both the Bushlo6 and Z a f f a r ~ n i ~ ~
type of chromatographic systems have been used for
hydrocortisone,and a typical sam ling is presented
in Table 4 according to Neher. o y Additional
systems can be found in references 108, 109, and
110. The use of fully acetylated paper,lll paper
impregnated with stearato chromic chloride for
reverse phase as well as centri-
fugal acceleration113has been applied to hydro-
cortisone.
HYDROCORTISONE 301

TABLE 4
Paper Chromatographic Systemslo7
Zaffaroni Type Systems:
Propylene/toluene 0.01
Formamide/chloroform 0.24
Formamide/ethyl acetate-butyl acetate-
water (15:85:1) 0.40
Formamide/butyl acetate:water (100:5) 0.42
Formamide/n-butanol-butyl acetate-
water (15:85:5) 0.60
Bush Type Systems:
Hexane-tert. butanol-methanol-water 0.19
Benzene-methanol-water (100:50:50) 0.25
Cyclohexane-dioxane-methanol-water
(4:4:2:1) 0.25
Toluene-ethyl acetate-methanol-
water (9O:lO: 50: 50) 0.37

For detection and quantitation,

ultraviolet light,ll1,ll4 blue tetrazolium,77
diphenylstyryl phenyltetrazolium chloride,l16 and
alkaline fluorescence117 have been used. Quantita-
tion of hydrocortisone by direct photometry of
paper chromatograms, using an adapter in a Beckman
DU spectrophotometer has been reported.ll* For
preparative paper chromatography to separate hydro-
cortisone from other adrenal constituents, see
reference 119.
5.82 Thin-Layer
Thin-layer chromatography started to
be applied to hydrocortisone in 1960. The two
earliest publications specifically mentionin hydro-
cortisone seem to be those b Carr and ReddyqPo and
by Cerny, Joska and Labler. 1 x 1 TLC is still an
important method for hydrocortisone. Until 1981,
the USP assay32 for hydrocortisone was based on
quantitative TLC,and the BP33 uses TLC for the
related steroid test. For general references, also
concerning quantitation, see references 3 , 107 and
110. Some typical systems have been summarized in
Table 5. While ultraviolet, blue tetrazolium and
sulfuric acid are employed as detection agents in
 TABLE 5

SUPPORT SOLVENT SYSTEM Rf DETECTION REF.

-
Cellulose acetate Water-tert.butano1-pet.ether (2:1:3) - Fluorometric 120
Silica Gel Benzene-ethyl acetate (1:l) 0.22-0.36 Sulfuric acid 121
Celite Formamide/benzene-chloroform 0.13 Sulfuric acid, 125
(1:l) blue tetrazolium,
isonicotinic acid,
hydrazide
Silica Gel G Chloroform-methanol-water (188:12 :1) Sulfuric acid 126
Ethyl acetate-chloroform-water Ultraviolet
(90 :10 :1)
Silica Gel G Hexane-ethyl acetate (1:1) 0.06 Phosphomo1ybdic 127
(starch bound) Ethyl acetate 0.38 acid (10%in
Benzene-isopropanol (4:1) 0.55 ethanol)
Alumina
(acetic acid treated) Benzene-dioxane ( 2 : l ) 0.25 Ultraviolet 128
Benzene-dimethylformamide (9:1) 0.53
Chloroform-ethanol (96:4) 0.35
Diisopropylether 0.25
Silica Gel G Dichloroethane-methyl acetate- Diphenyl (styryl- 129
water (2:l:l) phenyl)-tetrazolium
Silica Gel G Chloroform 0.43 Toluene sulfonic acid 130
(impregnated with Methylene chloride-toluene (1:1! 0.10
formamide
 TABLE 5 (Cont'd)

SUPPORT SOLVENT SYSTEM Rf

- DETECTION -
REF.

polyamide Resin Chloroform-ethanol (97:3) 0.50 Blue tetrazolium 131

Methylene chloride 0.23

Silica Gel G Cyclohexane-ethyl acetate- 0.23 Potassium periodate 132

ethanol (4.5 :4.5 :1) formaldehydog enic "
I'

Chloroform-ethanol (9:1) 0.18

Ethyl acetate-n-hexane-acetic
acid-ethanol (72:13.5:10:4.5) 0.44
Benzene-ethanol (4:1) 0.37
Silica Gel Butyl acetate-methanol (99:l) 0.48 Ultraviolet 107
W
W
0 Chloroform-methanol (9:1) 0.42 Ultraviolet
304 KLAUS FLOREY

most cases, Lisboa122 has described quite a number

of other "in situ" color reactions. Attention has
also been paid to the elution and uantitation of
steroids after chromatography. I g2 I Nanogram
amounts have been determined, and best recovery
was achieved by elution with methan01.l~~
A system for the separation of
hydrocortisone from its 17-keto oxidation product
has been described.133 Densitometry has also been
used for quantitation.134,135r136
5.83 Column
In the early years, column and
partition chromatography was used extensively for
the separation of hydrocortisone from adrenal gland
mixtures138 and the determination in blood.lol For
general reviews, see references 2, 3 and 119.
Diatomaceous earth (celite, superce1)101r138r139
silica ge1,140-143sephadex LH-20,144 G-50145 and
diatomaceous earth treated with a ~ e t o n i t r i l e l ~ ~
have been used as adsorbents with various solvent
mixtures.
5.84 High Performance Liquid
Chromatography (HPLC)
HPLC has swept the field of
pharmaceutical analysis. Shall wonder then that
HPLC is also rapidly replacing older chromato-
graphic methods for the control of the purity of
hydrocortisone and for its determination in form-
ulation and biological fluids (see section 8 and 9).
General reviews can be found in reviews by
Vestergaard,2 Di,! Smith,147 O'Hare and Nice,147
and Gorog and Szasz.3 The first reported use of
HPLC for h drocortisone seems to be by Siggia and
Dishman. 1 4 g Some typical data are summarized in
Table 6. A proposed selected method for the
analysis of hydrocortisone in serum has been
described.149
5.85 Gas Chromatography
A description of early attempts on
gas chromatography of hydrocortisone-can be-found ,
in the book b Wotiz and Clark.163 VandenHeuvel
and Horning16< already found in 1960 that gas
chromatography of underivatized hydrocortisone and
other steroids with the dihydroxy acetone side
 TABLE 6
HPLC SYSTEMS

MOBILE PHASE COLUMN DETECTION -

REF.

Water and methanol, water (3:7) Trifluoroethylene beads coated Ultraviolet 148
with Amberlite LA-1
Ethyl acetate ( 5 % ), acetonitrile Zipax coated with oxydipropionitrile Ultraviolet 150
(0.2%) in hexane
1%Methanol in water Cyano ethyl silicone Ultraviolet 151

Chloroform, dioxane (20:l) Silica gel Ultraviolet 152

Ethylene chloride, ethanol, water Zorbax-Sil Ultraviolet 153

Aqueous ethanol with tetrapentyl Silica coated with octadecyl silane Ultraviolet 154
ammonium chloride
1.5% Methanol and 0.2% water Silica gel Ultraviolet 155
in chloroform
Methanol, water, acetic acid c18 Bondapak Ultraviolet 156
(53:42: 5)
Methylene chloride, methanol, Silica gel Ultraviolet 157
tetrahydrofuran, acetic acid
Acetonitrile, water, glacial ODS Ultraviolet 158
acetic acid (38:60:2)
Methanol-O.OlM ammonium acetate c18 Bondapak (gradient elution) Ultraviolet 159
pH 6.9 (10:90)
60% aqueous methanol c18 Lichrosorb Ultraviolet 160
 TABLE 6 (Cont'd)

MOBILE PHASE COLUMN DETECTION REF.

-
*Ethylene dichloride, butanol, water Fluorescence 161
(91: 8 . 5 : 0.5)
Butyl chloride, tetrahydrofuran, s lica gel Ultraviolet 33
methanol, glacial acetic acid
(190:14:7:6)
Acetonitrile, methanol, water Silica coated with octadecylsilane Ultraviolet 33
(2:2:6)
Acetonitrile (20%) in 0.01M Lichrosorb RP-8 Ultraviolet 162
phosphate buffer pH 6 containing
0.01M tetrabutyl ammonium bromide
w
0
ch
dansyl hydrazine derivative

chain eliminated the side chain during vaporization and that the retention time
was that of the corresponding 17-keto compound. The main use of gas chromato-
graphy has been found in the determination of hydrocortisone in biological
fluids where it is competing with radioimmunoassays. Recently, it has been
coupled with mass spectrometry.165 GC/MS and RIA methods have been
Some typical data are presented in Table 7.
5.9 Electrophoretic
Hydrocortisone, being neutral, should not be moved by electrophoresis.
However, there is a report that hydrocortisone migrated in pH 7.0 phosphate buff-
er and pH 10 bicarbonate buffer when subjected to high voltage electroph~resis?~~
 TABLE 7
GAS CHROMATOGRAPHIC SYSTEM
COLUMN
DERIVATIZATION COLUMN PACKINGS TEMP. DETECTOR -
REF.
- Silicone SE-30 on 222 Flame ionization 164
Chromosorb W
Trimethylsilyl ether 3% dimethylpolysiloxane - Flame ionization 167
on Gas Chrom. P
Methoxime-trimethylsilyl ether 1% SE-30 on Gas Chrom. P 250 Flame ionization 168
Methoxime-trimethylsilyl ether 3% OV-1 Diatomite CQ 240 Flame ionization 169
Periodate oxidation to SE-30 250 Flame ionization 170
w
eticholenic acid
0
rl llfksilylether and 17,21 1% OV-1 on Chromosorb G 240 Flame ionization 171
cyclic dimethyl
s iliconide
Trimethylsilyl ether-enol- 1% OV-1 on Gas Chrom. P 250 Flame ionization 172
trimethylsilyl ether
Chromium trioxi.de oxidation to 3% OV-17 on Chromosorb W 230 Electron capture 173
androstenetrione and forma-
tion of heptofluorobutyrate
3.20 Dimethoxime-11,17,21 tri- 3% SE-30 - Mass fragmentography 165
methylsilyl ether at m/e 636; m/e 638
(14C-hydrocortisone)
308 KLAUS FLOREY

5.10Bioassay-Enzymatic
The original workers, isolating hydro-
cortisone and other adrenal hormones from adrenal
tissue, used adrenalectomized animals and later on
such tests as the liver glycogen, e ~ s i n o p h i l , ~ ~ ~
thymus involution' and electrolyte-excret-
ing177 (cf. 1, 600ff). The enzyme 20-B-hydroxy
steroid dehydrogenase coupled with DPNH has also
been used to determine hydrocortisone in blood
plasma.178
5.11Saturation Analysis
The great importance of the accurate
determination of hydrocortisone body fluid levels
in normal and diseased states has led to the
development of the following techniques which can
be classified as saturation analysis.' Each of the
techniques has its proponents and pitfalls,179 but
at present, radioimmunoassay seems to be the most
useful.
5.111
Competitive Protein Binding
A review of this technique can be
found in the chapter by W. R. Slaunwhite, Jr. in
reference 2. Murphy first employed this technique
to study hydrocortisone levels in plasma and other
body fluids.lso,lB1 The competition of hydro-
cortisone in the plasma sample with added hydro-
cortisone-4-C14 for corticosteroid-binding globulin
(a1 fraction) is the basis of the method. The
method was extended to urine and the purification
prior to assay refined.l*' TLC has been used for
prepurification.183 Tritiated instead of carbon-14
labelled hydrocortisone has also been used.178
Horse transcortin was used to measure hydrocort-
isone in umbilical cord serum and amniotic f 1 ~ i d . l ~ ~
A commercial kit has been described.186 Ultra-
centrifugation was used to determine unbound
hydrocortisone.187
5.112 Double Isotope Derivative Assay
The double isotope derivative
assay is a modification of competitive protein
binding analysis where a trace amount of tritiated
hydrocortisone is added to determine recovery
values durin extraction. In a comparison of the
two methods,q 8 8 it was found that generally there
is good agreement; however, a large discrepancy
HYDROCORTISONE 309

was found in the values obtained in newborns and

patients with adrenogenital syndrome, where
competitive protein binding gives much higher
values, probably due to the presence of related
steroids competing with hydrocortisone.
5.113Radioimmunoassa
Ruder, Guy and ;ipsettlsg are
credited with the first description of a radio-
immunoassay for hydrocortisone in 1972, but others
were also hard at work. That this technique
offered advantages as to higher sensitivity and
greater specificity over protein-binding methods is
attested by the mushrooming literature which here
is reviewed very selectively. Brief reviews are
those by G. E. Abraham in references 2 and 192; a
detailed review is that of P. Vecsei in reference
193. Antigens used for immunization were the 21-
hemisuccinate, the 3-oxime-21-acetate, the 3-oxime,
the 3,20-dioxime and the 6a- or 66-hemisuccinoxy
derivatives to obtain antibody titers in sheep and
rabbits. These antibodies had varying percenta es
of cross reactivity to related steroids. 3H, 7zSe
and 125I-ligands are used, but in one comparison
study,lq4 tritiated hydrocortisone was found to
have the best sensitivity. Most laboratories use
dextran coated charcoal to separate protein-bound
and unbound radioactivity. The usual solvents for
extraction of plasma hydrocortisone are alcohol
and dichloromethane. Comparison of the radioimmuno-
assay with chemical ionization/mass spectrometry166
and with HPLCIq5 have been reported. An inter-
laboratory evaluation of four RIA kits has been
made.lg6 An automated assay method has been
evaluated.
5.114Other
A chemiluminescent immunoassaylg8
has been described. Enzyme-labelled immunoassays,
using alkaline phosphataselg9 and E. coli B-galac-
tosidase200 have also been developed.
6. Stability - Degradation
Hydrocortisone is very stable as a solid. In
aqueous and alcoholic solution, the dihydroxy-
acetone side chain, as in all such corticosteroids,
is prone to oxidative rearrangement and degradation
at very acid and particularly also alkaline pH's.
310 KLAUS FLOREY

For instance, it was reported in 1967201 that

hydro~ortisone-4-~~C in aqueous or methanolic
solution decomposed to products of lesser and high-
er polarity. The main decomposition product was
identified as 118-h droxyandrost-4-ene-3,17 dione.
In another study,20Y it was found that aqueous
solutions of hydrocortisone were spontaneously
oxidized to 21-dehydrocortisol and other products.
In pH 9.1 carbonate buffer, the conversion rate was
1.6-2.8%/hr. Addition of EDTA or high concentra-
tions of plasma or plasma protein fractions slowed
the degradation.
Recently, Hansen and Bundgaard162 have studied
the degradation of hydrocortisone in aqueous
solution in detail. They identified degradation
products by HPLC (see Section 5.84) and treated the
data kinetically. They confirmed results of
previous investigators, partially obtained with
other corticosteroids with a dihydroxyacetone side
chain. They identified the glycolic acid V, not
previously reported. Generally, they found
degradation patterns to be more complex in basic
solutions. Their results are summarized in
Figure 6.
The photolytic degradation of hydrocortisone in
alcoholic solutions when exposed to fluorescent
lighting was found to be first order. The half-
life of such a solution at room temperature was
found to be 160 days. The degradation involved the
A-ring of the molecule, as measured by a decrease
in U.V. absorption, but not the side chain as
determined by blue tetrazolium. No degradation
products were described.203
7. Metabolic Products - Pharmacokinetics
The metabolism of hydrocortisone is complicated
since it can be different both qualitatively and
quantitatively in health or disease. Much pioneer-
ing work has been done with and without the use of
14C-labeled hydrocortisone in isolated liver204 and
kidney205 in intact rats,206 and in man.207,208
However, the definitive quantitation study in
normal man is that of Fukushima, Bradlow, Hellman,
Zumoff and Gallagher,209 which is summarized in
Figure 7. It was found that 9 0 % of the radio-
activity in the neutral steroid extract would be
HYDROCORTISONE 311

HC=O c=o
I I
c=o

YT-
C-OH

IX IV
(minor) (major)

21 C H 2 0 H
I IV
20 c=o (minor)

c=o
I
c=o

b'"
I11

I11
COOH
I (intermediate)
I
I
0 CHOH

+ y1 + others
C CHOH
VIII VI . ..OH
(major) (major) (minor) + others

(major) (major) (minor)

Fig. 6. Degradation pathways of hydrocortisone in aqueous

solution. The numbering is that of reference 162.
All oxidative pathways in neutral and alkaline
solution are metal (copper) catalyzed and can be
blocked by EDTA.
312 KLAUS FLOREY

CH20H CH20H
I I
c-0 c-0

68-Hydroxyhydrocortisone Cortisone

CH20H
l
Hydrocortisone

HO”

8
Hd
R-OH Tetrahydrocortisol; Allotetrahydrocortisol
R-0 Tetrahydrocortisonei Rllotetrahydrocortisona

CH20H
I OH HO&.
CHOH

’ __+

o*
HO‘ H HO’
li
- -- cortol -- allocortol
J
20 o+R Dihydrocortisol R-OH 20 o+B 20 o+E
R=O 20 a+B cortolone 20 a+B allocortolone

1
HO‘ @
116-Hydroxyetiocholanolone
+ HA .(y.p
;I
118-hydroxy androsterone
Gluconurides

Figure 7. Metabolic Pathways of Hydrocortisone.

HYDROCORTISONE 313

accounted for by the compounds depicted in Fig. 7,

and 70% were recovered in urine as the glucuronides.
By far, the largest fractions were the tetrahydro
derivatives both of hydrocortisone and cortisone
with the side chain intact, followed by the
cortolones and cortols. The formation of 6B-
hydroxycortisone and 17-keto compounds was found
to be minor.
Recently, the pharmacokinetics of orall
The
administered hydrocortisone was described. l X 0
mean elimination half-life of hydrocortisone from
plasma was found to be about 9 0 minutes.
Detailed reviews on the subject are available
available. I 210 I 2 2 0
8. Determination in Biological Fluids and Tissues
Since hydrocortisone is a hormone, determina-
tion in bioiogical fluids and tissues-has been of
particular importance, starting with the animal
assays leading to its isolation (cf. ref. 1,
600ff). A perusal of the general reviews,147
I I I mentioned in the preceding pages
may be useful. The following is a compilation of
references, most of them grouped according to
fluid or tissue:
Blood (Plasma, Serum) :
Porter-Silber: 7 5 , 7 6
Fluorometric: 91, 1 4 2 , 2 1 1 , 212
Paper Chromatography: 1 0 6 , 117
Polarographic: 1 0 1
HPLC: 1 6 1 , 1 4 9 , 1 5 7 , 1 5 6 , 1 5 3
Gas Chromatography: 173
Gas Chromatography/Mass Spectrometry:
165, 198
Competetive Protein Binding: 1 7 9 , 180,
181, 183, 186, 187, 219
Radioimmunoassay: 1 6 0 , 1 6 6 , 1 8 9 , 197
Chemiluminescent 1 A : 1 9 8
Enzyme 1 A : 1 9 9 , 2 0 0
Enzymatic: 1 7 8
Urine :
Porter-Silber: 74
Fluorometric: 213
314 KLAUS FLOREY

Paper Chromatography: 109, 207, 209, 214

TLC: 215, 216
HPLC: 155
Compet. Protein Binding: 181, 182, 184
Radioimmunoassay: 195
Reverse Isotope Dilution: 209
Adrenals :
Biological assays: cf. 1, p. 600ff
Paper Chromatography: 118, 217
Amniotic Fluid:
Compet. Protein Binding: 185
Liquid Chromatography: 40
Milk :
TLC: 137
Cerebral Spinal Fluid:
Compet. Protein Binding: 181
9. Determination in Pharmaceutical Formulation
The following tabulation highlights the methods
referenced previously which have been used in the
assay of pharmaceutical formulations:
Tablets:
Spectrophotometric (isonicotinic acid
hydrazide) : 78
Automated colorimetric: 79, 86, 176
Spectrophotometric (ammonium molybdate); 87
Tetrazolium: 33, 115
Paper Chromatography: 115
TLC: 33
Column: 146
HPLC: 33
Topicals:
Blue Tetrazolium: 33
Spectrophotometric (cupric acid ox.): 81
Polarography: 99, 102
TLC: 124, 136
Column Partition: 138
HPLC: 33, 147, 150
HYDROCORTISONE 315

Parenteral:
Blue Tetrazolium: 33
HPLC: 1 5 8

References
1. L.F. Fieser and M. Fieser, Steroids, Reinhold
Publishing Corporation ( 1 9 5 9 ) .
2. Modern Methods of Steroid Analysis,
E. Heftman, ed., Academic Press ( 1 9 7 3 ) .
3. I.S. G6rog and G . Sza'sz, Analysis of Steroid
Hormone Drugs, Elsevier ( 1 9 7 8 ) .
4. T. Reichstein, Helv. Chim. Acta, - 20, 9 5 3
(1937).
5. P.S. Hench, E.C. Kendall, C.H. Slocomb and
H.F. Polley, Proc. Staff Meetings, Mayo Clinic,
-
24, 1 8 1 (1949).
6. T. Reichstein and C.W. Shoppee, Vitamins and
Hormones, 1, 3 4 5 ( 1 9 4 3 ) .
7. N. L. Wendier, R.P. Graeber, R.E. Jones and
M. Tishler, J. Am. Chem. SOC., - 72, 5 7 9 3 ( 1 9 5 0 ) .
8. L.H. Sarett, J. Am. Chem. SOC., - 70, 1 4 5 4 (1948).
9. K. Florey, Chimia, 8, 8 1 ( 1 9 5 4 ) .
10. S.H. Eppstein, P.D.-Meister, H.C. Murray and
D.H. Peterson, Vitamins and Hormones XIV, 3 5 9 ,
Academic Press ( 1 9 5 6 ) .
11. M. Sittig, Pharmaceutical Manufacturing
Encyclopedia, Noyes Data Corp. ( 1 9 7 9 ) .
12. D. Riad-Fahmy, G. Read and I.A. Hughes,
Hormones in Blood, Vol. 3, p. 1 8 0 , C.H. Gray
and V.H.T. James, editors, Academic Press
(1979).
13. 0. Hechter and G . Pincus, Physiol. Rev., -
34,
459 (1954).
14. L.T. Samuels and T. Uchikawa in Adrenal Cortex,
p. 6 1 , Little, Brown and Co. , Boston ( 1 9 6 7 ) .
15. E. Ileftmann, Steroid Biochemistry, Academic
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