21.1.

03
AOAC Official Surplus Method 946.05i
i
BetaEstradiol in Drugs
Colorimetric Method

First Action
Surplus 1970
36.249
Apparatus
(a) Spectrophhotometer or photometer.See
955.55i
i(a).
(b) Sepearators.See 955.55i
i(b).
(c) Glass-stoppered test tubes.18 150 mm.
(d) Burets.Stopcocks lubricated only with reagent.
Orifice of one buret should be enlarded, if necessary, to
deliver 1 mL Reagent A in 30 sec or less. Protect
reagents in burents from moisture with suitable guard
tubes.
(e) Chromatographic tube.Select 25 200 mm test
tube of 3.854.00 sq cm crosssectional area by
measuring ht of 50 mL column of H2O in it. Fuse 6 cm
length of 56 mm tubing to bottom of tube and slightly
constrict this stem ca 2 cm below seal.
(f) Benzene reservoir.500 mL separator with 3 mm
or larger bore stopcock lubricated only with H2O. Stem
should be ca 10 cm long.
(g) Packing rod.Flatten end of glass rod to circular
head to provide clearance of ca 1 mm in chromatgc
tube.
(h) Leveling rod.Sharp-edged rod ca 1.5 cm diam.
36.250
Reagents
(a) Reagent A (stock iron-Kober reagent).Dissolve
1.054 g FeSO46H2O (Mohr salt) in ca 20 mL H 2O; add
1 mL H2SO4 and 1 mL 30% 2H 2O . Mix, heat until
effervescence ceases, and dil. to exactly 50 mL. To 3
vols of the Fe soln in vol. flask add H2SO4, with
cooling, to make 100 vols.
Redistill phenol, discarding first 10% and last 5%.
(Caution: Phenol may be harmful. Avoid Contact with
skin and eyes and breathing vapors.) Collect distillate
with exclusion of moisture in dry, tared g-s flask of ca
twice vol. of the phenol. Place stoppered flask in ice
bath to solidify phenol, breaking top crust with glass
rod to ensure complete crystn. Dry and weigh flask.
Add to phenol 1.13 times its wt of Fe-H2SO4 soln,
stopper flask, and let stand without cooling but with
occasional mixing until phenol is liquefied (ca 30 min
or less). Shake mixt. vigorously until homogenous and
let stand in dark 1624 hr. Add to mixt. 23.5% its wt of
H2SO4 (10+11). Shake vigorously to homogeneity.
Transfer to dry g-s bottles. Stored in dark and protected
from absorption of moisture, this reagent is stable for
months.

(b) Reagent B.To measured vol. Reagent A in g-s

graduated cylinder add 0.45 vol. H2O, mix, cool, and
transfer to dry g-s bottles. Store in dark and protect
from absorption of moisture. Inspect before use and
disperse any disperse any flocculent ppt by vigorous
swirling of reagent. With this precaution, reagent may
be used satisfactorily for weeks.
(c) Reagent C.To carefully measured vol. Reagent
A in g-s graduated cylinder add 0.45 vol. 1N HCl, mix
vigorously, and place at once in H2O bath at 2528E.
Use reagent preferably 1 hr and #3 hr after prepn.
(d) Packing material.Celite 545 (Johns-Manville
diat. earth).
(e) Sodium hydroxide soln.0.400N (carbonatefree).
(f) Benzene.Reagent grade, redistd in all-glass app.
36.251
Standard Solutions
(a) Beta-estradiol std soln.20 Fg/mL. Dissolve
exactly 10 mg pure $-estradiol in alcohol and dil. with
alcohol to 100 mL in vol. flask. Pipet 10 mL of this soln
into 50 mL vol. flask and dil. to vol. with alcohol.
(b) Alpha-estradiol std soln.20Fg/mL. Prep. as in
(a) from pure "-estradiol.
(c) Beta-dihydroequilin std soln.10 Fg/mL. Prep.
as in (a) from 5 mg pure $-dihydroequilin.
Stored in dark in tightly stoppered containers, std
solns keep for months.
36.252
Preliminary Determination
Apply Methods A and B, 955.55i
i, directly to
aliquots of CHCl3 soln of diols obtained from 955.55i
i
(Very turbid solns sometimes resulting from these
aliquots should be filtered, along with blank and std
solns, thru pledget of fine glass wool packed tightly in
lower end of stem of funnel.) Calc. $-estradiol content,
disregarding presence of $-dihydroequilin. If wt $estradiol so detd is #1% of wt Ketosteroids, report $estradiol as Not >1% of ketosteroid content. If $estradiol content is >1% of wt ketosteroids, repeat detn
on aliquot of the CHCl3 soln, using chromatge sepn,
955.55i
i.
36.253

Preparation of Chromatographic
Column
(Caution: See Appendix B., flammable solvents,
toxic solvents, benzene.)

Pack fine glass wool into constricted stem of

chromatgc tube so that when tube is filled with
benzene, rate of flow is 2.53.0 mL/min. Before
packing column, fasten piece of rubber tubing with
attached screw-clamp to outlet to control flow during
packing.

2
Cover 1 g Celite in small beaker with benzene. Pipet
in 0.5 mL H2O and mix vigorously with stirring rod
until Celite is uniformly wet. With tube ca filled with
benzene, place pad of glass wool (ca 1 cm high when
gently compressed) at bottom of tube and then transfer
Celite mixt. to tube.Form flocculent suspension by
slowly working packing rod up and down as piston thru
Celite mixt. Gently compress Celite with packing rod
and finish off edges with leveling rod to form level,
shorply defined surpace on uniform pack ca 1 cm high.
Cover 8 g Celite in mortar with ca 40 mL benzene
and distribute exactly 5 mL 0.400N NaOH over Celite
from pipet. Carefully mix several min with pestle until
Celite appears uniformly wet. Open screw-clamp
enough to permit slow drainage during packing of tube.
Transfer Celite to tube with spatula in ca 5 portions,
suspending each portion and gently packing as above.
Finish off top of column, scraping down any Celite on
upper wall of tube to form sharply defined, level
surface on column of ca 30 mL vol. over initial pack.
(Packing too tightly may cause loss of estradiol in
forerune, particularly at room temp. much >25E. Celite
must be covered with benzene at all times.)
Nearly fill reservoir with benzene and seal stopper
with film of H2O to prevent air leaks. Insert stem of
funel into benzene over the Celite, open stopcock fully,
and adjust level of benzene to produce flow rate of
2.02.5 mL/min with screw-clampfully open. Mark
level of benzene on tube, close stopcock, and remove
benzene reservoir.
36.254
Determination
(Caution: See Appendix B., flammable solvents,
toxic solvents, benzene.)
Carefully evap., to just short of dryness, aliquot
CHCl3 ext, 955.55i, contg 100250 Fg total diols
calcd as $-estradiol. If necessary, add few mL alcohol
near end of evapn to help remove any residual H2O.
Remove last portions of solv. in efficient desiccator
connected to vac. for $1 hr.
Dissolve dry ext in 5 mL benzene by warming gently;
then cool soln to room temp. or below. Remove rubber
tubing from partition tube and when benzene just stops
dropping from tube, transfer diol soln at once to tube,
letting it flow down wall near top of Celite (5 mL pipet
is convenient for this purpose). When benzene just
stops dropping from tube, complete transfer in like
manger with 3 addnl 5 mL portions benzene, discarding
effluent. Immediately place 50 mL graduated cylinder
under tube, add benzene to level marked on tube, and
replace benzene reservoir, supporting it at ht to
maintain that level when stopcock is fully opened.
When 30 mL effluent collects in cylinder, replace

cylinder as receiver with dry 250 mL beaker previously

marked at 170 mL level. (Decrease 30 mL forerun by 2
mL for each 1E that room temp. is >25E.) Collect 170
mL effuent in beaker, conc. soln to 3040 mL, transfer
to 50 mL vol. flask, and dil. to vol.
Det. $-estradiol in soln by Methods A, B, and, if
necessary, C.
Method A.Transfer to dry 18 150 mm g-s test
tubes: (1) Aliquot of sample soln contg 1025 Fg total
estradiols; (2) 1 mL "-estradiol std; (3) 1 mL $estradiol std; and (4) if necessary from Method B, 1 mL
$-dihydroequilin std (see Note). Add several pieces of
SiC, 955.55i
i(h), to each tube and rapidly evap. solv.
in steam bath (do not use air current) until ebullition
from boiling stones just stops. Instantly remove tube,
quickly wipe dry, and transfer to efficient vac.
desiccator. Keep connected to vac. $1 hr.
To each tube and to blank tube add glass bead, and
measure 1 mL Reagent A into each tube from buret,
quickly wiping outside of tip with piece of absorbent
paper before each addn. Stopper immediately and let
stand 30 min, vigorously shaking tubes at 5 min
intervals. Place in boiling H2O bath 35 min, removing
and shaking each tube few sec after first 5 min.
Transfer to ice bath 2 min; then remove and add exactly
4 mL H2SO4 (7+13) from buret. Let stand 5 min; then
mix by shaking, first gently, then vigorously, to
homogeneity.
Measure A of sample and stds relative to blank at 525
nm (midpoint between max. for "-estradiol and $estradiol) and at 420 nm, making any necessary
corrections for cell variations.
Fg total estradiols in aliquot (calcd as $-estradiol)
= Ta = 20
A525 nm (sample) ! [(A420 nm (sample))/2]
A525 nm($-estradiol std) ! [(A420 nm ($-estradiol std))/2]

g "-estradiol in aliquot (calcd as $-estradiol) = Ba =

Fg "-estradiol in aliquot (from Method B)

A525 nm ("-estradiol std) ! [(A420 nm ("-estradiol std))/2]

A525 nm ($-estradiol std) ![(A420 nm ($-estradiol std))/2]

Fg $-dihydroequilin (DHQ) in aliquot (calcd as $estradiol)

= Da = Fg DHQ in aliquot (from Method C)

2A525 nm (DHQ std) ! A420 nm (DHQ std)

A525 nm($-estradiol std) ! [(A420 nm ($-estradiol std))/2]

3
Fg $-estradiol in aliquot = Ta ! (Ba or Da or both),
depending on composition of aliquot.
Note: If $-dihydroequilin in aliquot is >10 Fg, use
correspondingly higher std in detg correction for $dihydroequilin and correct calcn accordingly.
Method B.Transfer to dry 18 150 mm g-s test
tubes: (1) Aliquot of sample soln contg 1025 Fg total
estradiols; (2) 1 mL "-estradiol std; and (3) 1 mL $dihydroequilin std (see Note). Add several pieces of
SiC to each tube, evap. solv., and dry residue as in
Method A. To each tube and to blank tube, add 1 mL
Reagent B from buret, quickly wiping outside of tip
with absorbent paper before each addn. Stopper
immediately and place in boiling H2O bath exactly 2
min, shaking tube after 30 sec for few sec without
removing from bath. Transfer to ice bath 2 min; then
remove and add from buret exactly 4 mL H2SO4 (7+13).
Mix by shaking vigorously to homogeneity. Promptly
measure A of sample and std relative to blank at 526 nm
(max. for "-estradiol), at 468 nm (max. for $dihydroequilin), and 420 nm, making any necessary
corrections due to cell variation. If
A468 nm (sample)
A
! A526 nm (sample) 458 nm ("-estradiol std)

A526 nm ("-estradiol std)

does not exceed 20% of A468 nm ($-dihyrdoequilin std), then
disregarding presence of $-dihydroequilin in like
aliquot will result in #0.8 Fg apparent $-estradiol in
Method A. This is normally negligible quantity, in
which case calc. "-estradiol as follows and omit
dihydroequilin std in Method A:
Fg "-estradiol in aliquot = 20
A526 nm(sample) ! (A420 nm (sample)/2)

A526 nm ("-estradiol std) ! (A420 nm ("-estradiol std)/2)

Note: If $-dihydroequilin in aliquot is >10 Fg, use
correspondingly higher std in detg correction for $dihydroequilin and correct calcn accordingly.
Method C.Transfer to 18 150 mm g-s test
tubes: (1) Aliquot of sample soln contg #20 Fg $dihydroequilin; and (2) 2 mL $-dihydroequilin std.
Add several pieces of SiC to each tube, evap. solv.,
an ddry residue as in Method A. Place tubes in H2O
bath at 2528E and rapidly add 5 mL Reagent C near
bottom of each. Using long stirring rod, mix residue
vigorously with reagent at least 1 min. Leave rod in
tube. Measure A of sample and std relative to reagent
(also held in bath at 2528E) at 472 nm just 30 min
after addn of reagent, and repeat measurement at

510 min intervals until max. A is reached (usually

3555 min after mixing).
g $-dihydroequilin in aliquot = 20 A472 nm (sample) /
A472 nm (std).
Reference: JAOAC 34, 58(1951); 37, 702(1954);,J.
Am. Pharm. Assoc., Sci. Ed., 35, 176(1946); 39, 37,
544(1950); 42, 167(1953(.
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