this topic is for analytical methods for determination of steroids

1. PRESENTED BY:
R.VENKATESH
Maharajahs college of
pharmacy

2. • Introduction
• Origin of steroids
• Classification
• Methods for determination of steroids
1 HPLC
2 UV spectroscopy
3 Visible spectroscopy
4 Infra Red spectroscopy
5 Thin Layer Chromatography

3. •A Steroid is an organic compound with four rings
arranged in a specific molecular configuration .
Example : lipid cholesterol , estradiol etc.
•A Steroid core structure is composed of 17 carbon atoms
bonded in 4 “fused” rings (3 six membered rings A B C
are cyclohexane rings & 1 five membered ring D is
cyclopentane ring) .
• Steroids vary by the functional groups attached to this
four –ring core and by the oxidation state of the rings .

4. A B
C D1
2
3
4
5
6
7
8
910
11
12
13
14
15
16
17
Cyclopentano-perhydro phenanthrene nucleus

5. • Steroids are found in plants , animals and fungi .
• Steroids are manufactured in cells from the sterols.
lanosterol ( from animals and fungi )
cycloartenol (from plants )
• The steroids which are from natural are originated
from adrenal cortex ,corpus luteum , ovaries , liver
and testes .

7. • HPLC
• Thin Layer Chromatography
• UV Spectroscopy
• Visible Spectroscopy
• Infra Red Spectroscopy

8. Steroids are analyzed by different analytical
methods. They are as follows:
a) Colorimetric methods
i) Reaction with tetrazolium salts
ii) Reaction with phenyl hydrazine
iii) Reaction with diphenyl amine
iv) Reaction with hydroxylamine
v) Reaction with Girard’s reagent
vi) Reaction with Gibb’s reagent
b) UV methods
c) IR methods
d) Electro-analytical methods
e) Gravimetric methods
f) Flourimetric methods

9. a) Colorimetric methods:
• This method is based on the chemical reaction of the
functional group with that of the reagents.
i)Reaction with tetrazolium salts:
This method was proposed by Burton and Keutmann for
the detection of α-ketolic steroids by using the triphenyl
tetrazolium chloride salt.
This salt undergoes reduction reaction.
Method:
step:1
Drug iso-octane alcohol mixture
extracts evaporated residue taken in to three
20ml portions of hot alcohol and stirred gently for 3-5min
continuosly .
step:2
1ml resulting solution 9ml alcohol+1ml dil.
Tetramethyl ammonium hydroxide solution.
extracted
added

10. Step:3
Then 1ml of triphenyl tetrazolium chloride is added
stand for 20min in dark
blank is carried out
absorbance measured at490nm
(visible spectrometer)

11. N N
N N
+
NH N
N N
(H)
(O)

12. ii) Reaction with phenyl hydrazine:
• This method was proposed by Porter and Silber.
• Based on the reaction of 17,21- dihydroxy-20-keto group of
steroids with phenylhydrazine dil. Acidic medium yellow
coloured compound. H2SO4
Method:
1ml of Sample solution + 8ml phenyl hydrazine-sulphuric acid
reagent
Blank is carried out by taking alcohol+ 8ml dil. H2SO4 .
sample &blank solutions are immersed in water bath at 60°C
for 20min.
cooling
absorbance measured at 410nm

13. iii) Reaction with diphenyl amine:
•This reaction is proposed by Clark.
•Based on reaction of the 17,21-dihydroxy-11,20-diketo
with diphenyl amine violet chromogen measured
at 530nm.
Method:
Sample alcohol+ diphenyl amine measured at
530nm.
dissolved

14. vi) Reaction with hydroxyl amine:
•This method was proposed by Zaffaroni.
•Based on the reaction of steroid esters with hydroxyl
amine hydrochloric acid & ferric chloride
Purple color.
RCOOR* + H2NOH RCONHOH +R*OH
3 RCONHOH +FeCl2 (RCONHO)3Fe + 3HCl
presence
Absorbance is directly proportional to concentration.

15. v) Reaction with Girard’s reagent-T:
Method:
Sample + 10ml of 2N NaOH + dil. H2SO4
Cool the solution, add 25ml ether
Solution is taken in separating funnel
Ether layer is separated
5ml portion is taken & add to 10% w/v Na2CO3 solution
5ml portion of distilled water, then evaporated to dryness
Residue is dissolved in CHCl3+ 100mg of Girard’s reagent-T
+

16. 5ml glacial acetic acid
Boil &cool, adjust the pH to 6.5-7 by adding 2N NaOH
Extract with 3 successive portions of 15ml CHCl3
Discard CHCl3 & acidify the solution with dil. H2SO4 &
stand for 2hrs at room temperature
Measure at 513nm
(visible spectrometer)

17. O
H3C
H3C
H3C
+ (CH3)3NHCH2CONHNH2)Cl-
O
H3C
H3C
H3C
N
N+
CH3
CH3
CH3
Reaction:
17-Keinstemid Girard’s reagent T Girard’s derivative

18. vii) Reaction with Gibb’s reagent:
Commonly known as 2,6-dibromo quinone chloro imide.
Method:
Gibb’s reagent + steroids
Red pigment
Extracted with CHCl3
Measured at 570nm

19. O
Br Br
N Cl
HO
+
O
Br Br
N
OH
Gibb’s reagent Phenol
Indophenol

20. b) UV Method:
• Mainly based on reaction of steroids with appropriate solvents.
1. a) 95% Ethanol + cortisone 238nm
b) 95% Ethanol+ hydrocortisone acetate 243nm
c) 95% Ethanol+ desoxy cortico sterone acetate 241nm
d) 95% Ethanol+ hydro cortisone 242nm
2. Steroids containing α-keto acid have λmax at 281nm in
phosphate buffer.
3. HCl + steroids 254nm
4. CHCl3 + steroids 390nm
5. Inorganic / organic alkali solution + 3-keto steroids
373nm – 375nm
6. Semicarbazide acetate solution + methyl testosterone
241nm
7. H2SO4 + cholesterol 390nm.

21. c) IR Method:
• Mainly based on the absorption of the steroids in carbon
disulphide at near infrared region.
• Carol describe the IR method for the oestrone & oestradiol
using alcoholic CS2
Method:
Sample dissolved in CHCl3
residue dissolved in chloroformic CS2
Measured at 935cm¯1 using reagent blank
evaporated

22. d) Electro analytical Method:
• Based on the principle of Potentiometric method.
• This undergoes reduction of α,β- unsaturated carbonyl
group of steroids at the dropping mercury electrode.
• This was first observed by Eisenbrand & Picher.
1 1
2 2
Girard’s reagent-T
steroids half-wave
potential
-1.5to-1.6V
-1.23V
Alkaline
iso-
propanol
solution

23. e) Gravimetric Method:
• Methods are based on the direct gravimetric principle.
• The below method is used for the determination of Anhydro
hydroxy progesterone.
Method:
• Sample is weighed in a thimble of a micro soxhlet which is
extracted with petroleum ether for 4hrs.
•Extract is evaporated.
•Thimble is removed & the residual petroleum ether is allowed
to evaporate.
•Again the extract is carried out with CHCl3 for 4hrs dried &
evaporated to dryness.
•Then the obtain residue is dried at 105°C to a constant weight.

24. Other method:
•This method is mainly used for the determination of
testosterone propionate.
Sample petroleum ether alcohol mixture
dryness
Obtain residue + 3ml of semi-carbazide acetate solution
Cooled and add 10ml of iso octane solution with continous
stirring.
extracted
evaporated
Reflux condensation
for 2hrs

25. Contents in the flask are poured into mixture of ice water &
normal water
Flask is washed with water & 2ml portions of methanol
Contents are placed in refrigerator for 3hrs
Residue dried at 105°C to constant weight.

26. f) Flourimetric method:
•Method was first observed by Kober for the determination
of urinary & plasma estrogens.
Method:
Natural estrogens concentrated H2SO4
yellow green
fluorescents
•Above method was modified by Boscott that addition of
diluents such as acetic acid or propylene glycol to produce a
specific fluorescents for estriol and estra diode.
dissolved

27. Drugs :
• Hydrocortisone
• Hydrocortisone Acetate
• Desoxycortone Acetate
• Dexamethasone
• Progesterone
• Hydroxy progesterone Hexanoate
• Testosterone propionate
• Cortisone acetate
• Betamethasone
• Oestradiol Benzoate
• Ethinyloestradiol
• Prednisolone

28. CH2OH
C=O
OH
HO
O

29. Identification:
a) IR spectroscopy
b) By TLC.
Solvent mixture :
Chloroform : Methanol(90:10)
Mobile phase :
Dichloromethane : Ether : Methanol (77:15:8) in 1.2Volumes of H2O
Test solution:
Dissolve 0.25g of substance in 100ml of solvent mixture.
Reference solution:
0.25%w/v soln. of Hydrocortisone RS in solvent mixture.

30. Test :
Apply 2ul of each solution . Develop the chromatogram. Dry the
plate in air and examine under UV light at 254nm. Spray the plate
with Ethanolic sulphuric acid solution heat at 120°C for 10min and
then observe under UV light at 365nm.
Assay:
Dissolve 0.1g of substance in ethanol and make it up to 100ml
Dilute 2ml of the soln. to 100ml with ethanol
Measure the absorbance at 241.5nm
Calculate the content ofC21H30O5 taking 440 as the specific
absorbance at 241.5nm

31. CH2OH
C=O
OH
HO
O
CH3
CH3
H
H H

32. Identification:
a) IR spectroscopy.
b) By TLC.
Mobile phase :
Add1.2Volumes of water and 8Volumes of methanol to a mixture of
15Volumes of ether and 77Volumes of dichloromethane.
Test solution:
Dissolve 25mg of substance in methanol and dilute to 5ml with same
solvent. Use this solution to prepare test solution b, dilute 2ml of the
solution to 10ml with dichloromethane.
Reference solution:
Similar to that of test solution but instead of substance hydrocortisone
acetate RS solution is taken.

33. Test :
Apply each solution on the plate . Develop the chromatogram.
Dry the plate in air and examine under UV light at 254nm. Spray the
plate with Ethanolic sulphuric acid solution heat at 120o for 10min
and then observe under UV light at 365nm.
Assay:
Dissolve 0.1g of substance in ethanol and make it up to 100ml
Dilute 2ml of the soln. to 100ml with ethanol
Measure the absorbance at 241.5nm
Calculate the content ofC23H32O6 taking 395 as the specific
absorbance at 241.5nm

34. HO
O
O
H3C
CH3
H
H H
H
O
O CH3
O

35. Identification
a) By IR spectrophotometry.
b) By TLC.
Solvent mixture :
Acetone : formamide (90:10)
Mobile phase :
Chloroform
Test solution:
25mg of substance in 10ml of solvent mixture.
Reference solution:
25mg of Cortisone Acetate RS in 10ml of same solvent mixture.
Test :
Apply 2ul of each solution . Allow the MP to rise 12cm. Dry the
plate in current of warm air, allow the solvent to evaporate. Heat at
120o for 10min, cool and examine daylight and UV at 365nm.

36. Powdered 20 tablets
Equivalent to 10mg of cortisone acetate was taken
Add 50 ml of methanol
Dil. to 100 ml with water
Use supernatant liquid
Assay:
Shake & centrifuge
Shake 2min (ultrasound)
Reference solution:
Dilute 50 ml of a solution in methanol containing 0.02 per cent w/v each of
cortisone acetate RS and prednisolone to 100.0 ml with water.
Test solution:

37. Chromatographic system:
Column : Stainless steel 25cm X 4.6mm packed with
ocadecylsilane bounded to porous silane (such as
hypersilODS)
Mobile phase : Methanol (60%)
Flow rate : 1.5 ml/min
Spectrophotometer : 240 nm
Injection volume : 20µl
• Inject the test and reference solution.
• Calculate the content of C23H30O6

38. SPECTROPHOTOMETRY:
• Dissolve 0.1g in ethanol dilute to 100.0 ml with same solvent
Dilute 2.0ml of the solution to 100.0 ml with ethanol.
• Measure the absorbption maxima at about 237 nm.
• Calculate the content of C23H30O6 taking 395 as the specific
absorbance at 237 nm.

39. CH3
C=O
H3C
H3C
O

40. Identification:
a) IR spectrophotometry.
b) By TLC
Mobile phase :
Mixture of 66V dichloromethane and 33V ethyl acetate.
Test solution:
Dissolve 0.1g of substance in 100ml of 9v of dichloromethane and
1V of methanol.
Reference solution:
A 0.1% w/v solution of progesterone RS.
Test :
Apply each solution on the plate . After development, dry the
plate in air, then observe under UV light at 241nm.

41. Spectrophotometry:
50 mg of Progesterone + sufficient dichloromethane 100.0 ml.
Dilute 3.0 ml to 50.0 ml with dichloromethane.
To 5.0 ml of the solution + 10 ml of isoniazid solution + sufficient
methanol 20.0 ml. Stand for 45 minutes. Measure the
λmax at about 380 nm, using 5 ml of dichloromethane as blank.

42. O
O
H
O
H H

43. Identification:
a) IR spectrophotometry
b) Liquid Chromatography.
Test solution :
50mg of substance in methanol (50ml)
Reference Solution :
2mg of substance and 2mn of testosterone acetate RS in methanol(50ml)
Chromatographic system:
Column : Stainless steel , 25cm X 4.6mm packed with
octadecyl silane bounded to porous silica.
Mobile phase : water : methanol (20:80)
Flow rate : 1.5 ml/min
Spectrophotometer : 254 nm
Injection volume : 20µl
Calculate the content of C23H31FO6 in the tablets.

44. 25mg of substance in 250ml ethanol
Take 5 ml and Dilute to 50ml with ethanol
Measure the absorbance of the resulting solution at 241nm
Blank solution : Ethanol
Reference solution: 0.006% w/v solution of testosterone propionate RS in
ethanol
Calculate the content of C22H32O3 in the tablets.
Assay:
44

45. CH2OH
C=O
OH
HO
O
CH3
CH3
H
H H
H

46. Identification:
a) By IR absorption spectrophotometry.
b) By TLC, coating the plate with silica gel G.
Solvent mixture :
Acetone : formamide (90:10)
Mobile phase :
Chloroform
Test solution:
Dissolve 25mg of substance in 10ml of solvent mixture.
Reference solution:
Dissolve 25mg of Prednisolone RS in 10ml of same solvent mixture.
Test :
Apply 2ul of each solution . Allow the MP to rise 12cm. Dry the plate in
current of warm air, allow the solvent to evaporate. Heat at 120o for 10min,
cool and examine daylight and UV at 365nm.

47. b) By Liquid Chromatography.
Test solution :
Dissolve 25mg of substance in 2ml of tetrahydrofuran and dilute it to 10ml.
Reference Solution :
Dissolve 2mg of prednisolone RS in the MP and then dilute it to 100ml.
Chromatographic system:
Column : Stainless steel , 25cm X 4.6mm packed with
octadecyl silane bounded to porous silica.
Column temp. : 45o
Mobile phase : tetrahydrofuran : water (220:700 ml) made up to 100ml
with water.
Flow rate : 1 ml/min
Spectrophotometer : 254 nm
Injection volume : 20µl
Calculate the content of C21H28O5 in the tablets.

48. Assay:
Weigh about 0.1g of substance and dissolve in sufficient
ethanol to produce 100ml.
Dilute 2ml to 100ml with ethanol
Measure the absorbance at about 243.5nm
Calculate the content of Prednisolone at 243.5nm.

49. References:
1. Indian pharmacopeia - 2007, volume - I, II, III.
2. A text book of pharmaceutical analysis, - Takeru
Higuchi & Einar Brochmann.
3. Steroids quantitative analysis – D. Satish Kumar.
4. Pharmainfo.net