METHODS OF ANALYSISOF

DIURETICS

INTRODUCTION TO FUROSEMIDE

Structure:

Molecular formula : C12H11ClN2O5SMolecular weight : 330.77g/molAppearance : crystalline powderCo lour : slightly yellowOdor : odorlessTaste : tasteless

Solubility : soluble in water, chloroform, ether, acetone, methanol, dimethyl formamide and in alkali hydroxides

Category : high ceiling efficacy diuretic

OCH2

HOOC

O2SH2N

Cl

NH

METHODS OF ANALYSIS:

1. Titrimetric method

2. Column chromatography

3. Thin layer chromatography

4. Gas chromatography

5. High performance liquid chromatography

6. Ultraviolet spectroscopy

7. Colorimetric method

8. Infrared spectroscopy

TITRIMETRIC METHOD:

Principle: Acid-base titration

Reaction:

+ NaOH

NH

H2NO2S

COOH

O

COONa

H2NO2S

NH O

Procedure:0.5g of furosemide

dissolve

40ml Dimethyl formamide

bromothymol blue

Titrated with 0.1N NaOH

• End point is blue colour.

• Each ml of 0.1NaOH = 0.03308g of C12H11ClN2O5S

COLUMN CHROMATOGRAPHY:

Principle : Column adsorption chromatography

Column : Stainless-steel column(15cm x 4.5cm)

Adsorbent : Lichrosorb RP-18 or RP-8

Mobile phase : Various mobile phases

Sample : 20mcl of urine

Detection : U.V at 243nm

THIN LAYER CHROMATOGRAPHY:

Principle : Adsorption

Adsorbent : Kiesulghur 60

Solvent : Chloroform: ethyl acetate: formic acid(14:6:1)

Standard : 0.2 to 10mg of furosemide and its metabolite

Sample : 0.5ml of plasma with 1ml methanol is centrifuged, a 50µl of the aliquot of the supernatant liquid is used.

Drying time : 3hours

Spraying reagent : 10% citric acid in aqueous ethanediol(1:1)

HPLC:

Sl

No

Stationary phase

Mobile

phase

Flow rate Detection Retention time

1. Bondapack C18 corasil (1.8x2mm)

0.02M KCl-HCl buffer pH 2

0.9ml/min U.V at 280nm

3min

2. µ Bondapack C18 column (30x4mm)

10mM

(NH4)2HPO4 in 25% methanol

2ml/min U.V at 254nm

3min

3. Lichrosorb RP8

(5µ)

Methanol-0.02M, phosphate buffer

of pH 3.0(1:1)

1ml/min Flourimetric detection

3min

4. Column packed with ODS(5µ)

Methanol: water: acetic acid

(40:57:3)

1.45ml/min

Flourimetric detection

3min

ULTRAVIOLET SPECTROSCOPY:

Sample : Lasix (furosemide preparation)Solvent : WaterAbsorbance maxima : 228nmE1%

1cm : 945

COLORIMETRIC METHOD:

• Furosemide produces blue colour when it reacts with butylamine,carbonyl chloride and acetic acid in a medium of anhydrous methanol.

• Colour is measured at 570nm.

• It can also be detected by mixing a solution of p-dimethyl aminocinnamaldehyde in 65%H2SO4, containing FeCl3, diluting with ethanol and measuring the absorbance at 530nm.

INFRARED SPECTROSCOPY:

Frequency(cm-1) Type of vibration Assignment3350-3400 NH C-NH1671 C=O -COOH1576 NH -NH21322 -S=O -SO2582 Cl C-Cl

GAS CHROMATOGRAPHY:

Principle : Partition

Sample : Trimethyl derivative of furosemide

Carrier gas : Nitrogen

Column : Silanisedglass column(1.8x2mm) packed with 3% of JXR (supelco) on Gas-ChromQ(100-120mesh)

Temperature : 245oC

Detector : 63Ni electron-capture detector

Internal standard : Trimethyl furosemide

INTRODUCTION TO SPIRONOLACTONEStructure :

Molecular formula : C24H32O4S

Molecular weight : 416.59 g/mol

Appearance : Crystalline powder

Colour : Yellowish-white

Odor : Faint mercaptan

SCOCH3O

O

O

METHODS OF ANALYSIS:

1. Thin layer chromatography

2. Ultraviolet spectroscopy

3. Colorimetric method

4. Infrared spectroscopy

5. Flourimetric analysis

THIN LAYER CHROMATOGRAPHY:Example 1:Solvent system : Ethyl acetate(100%)Adsorbent : Silica gel GF(Woelm)Detection : Short wave U.V, : Spraying with 50%H2SO4, heat to 80oC for 10 minutes, observe under long U.V, : Spray with phosphomolybdic acid.Rf value : 0.53

Example 2:Solvent system : Benzene:Ethyl acetate:Methanol(73:25:2)Adsorbent : Silica gel GDetection : Spray with phosphomolybdic acid and heat at 80oC for 10 minutesRf value : 0.67

INFRARED SPECTROSCOPY:

Wave number (cm-1) Assignment 1775 5 -membered,lactone carbonyl 1670-1690 3- ketone,7-thioester carbonyl 1620 4,5-double bond

ULTRAVIOLET SPECTYROSCOPY:

COLORIMETRIC METHOD:

• Reaction of spironolactone with methanolic hydroxylamine hydrochloride and

ferric perchlorate yields a red ferric hydroxamate complex having an absorbance

at about 515nm.

• Colour is stable upto 2hrs.

• Isonicotinic acid hydrazide may be reacted with spironolactone in methanolic

solution,yielding a soluble yellow product having an absorbance at 575nm.

FLUORIMETRIC ANALYSIS:Spironolactone

Dethioacetylated under mild acid Or alkaline conditions

4,6-dienone,canrenone

In 62% sulphuric acid

Fluorescent trienone(excitation maximum at 483nm and an

emission maximum at 525nm)

AMILORIDEStructure :

.HCl.2H2O

Mol formula : C6H8ClN7O.HCl.2H2OMol weight : 302.12g/mlDescription : Yellow to greenish yellow crystalline powder which is odorless.Solubility : Freely soluble in Dimethylsulfoxide, sparingly soluble in water, very slightly in ethanol.Brand name : MIDAMOR

METHODS OF ANALYSIS :

• Non – aqueous titration

• Thin layer chromatography

• UV spectroscopy

• HPLC

• IR spectroscopy

1. Non – aqueous titration :Principle :• Substances which are either too weakly basic or too weakly acidic to give sharp end point in aqueous solutions are determined by non-aqueous method.

Procedure :• Dissolve 450mg of sample in 100ml of glacial acetic acid• Add 10ml of mercuric acetate.• Add 15ml of dioxane.• Titrate with 0.1N perchloric acid using crystal violet as indicator

until a blue color.• Blank determination is done to make necessary corrections.

Each ml 0.1N perchloric acid = 26.61gm of Amiloride hydrochloride.

HClO4 H++ ClO4

-

CH3COOH CH3COO- + H

+

HClO4 CH3COOH+ CH3COOH2+ + ClO4

-

CH3COO-+ CH3COOH2

+CH3COOH2

N

N

Cl CN

NH2 NH2

O

CNH2

NH2

.HCl.2H2O + CH3COOH2+

N

N

Cl CN

NH2 NH2

O

C

NH2

NH+

Amiloride HCl

1. Thin layer chromatography : Principle :

• Main principle is adsorption.• Adsorbent acts as stationary phase, • Mobile phase selection depend on elutropic series based upon polarity of solvents.

Amiloride hydrochloride is analyzed by Normal phase TLC on silica gel by employing two solvent system ;

AMILORIDE AMILORIDE HYDROHYDRO

CHLORIDECHLORIDE

Solvent Solvent

system system Sample spotSample spot Detection Detection

of spot of spot Rf Rf

valuevalue Sensitivity Sensitivity

1) 10% n-1) 10% n-propanol in propanol in chloroform.chloroform.

2) 3N aqueous 2) 3N aqueous ammonium ammonium hydroxide + hydroxide + tetrahydrofuratetrahydrofuran (4 : 30)n (4 : 30)

5μl of 1% 5μl of 1% aqueous aqueous soln of soln of drug.drug.

1μl of 0.1% 1μl of 0.1%

aqueousaqueous soln of soln of drug.drug.

By UV at By UV at 254nm or 254nm or 360nm.360nm.

0.70.7

0.01%0.01%

0.1 to 0.5%0.1 to 0.5%

UV spectroscopy :

Principle :• Any molecule has either η,π and σ or a combination of these

electrons.

• Bonding (σ and π) & non-bonding (η) electrons abosorb the

characteristic radiation and undergoes transition from ground

state to excited state.

• By this characterization absorption peaks are obtained.

• There by the nature of electrons present and hence the molecular

structure can be elucidated.

Procedure :• UV spectrum of amiloride can be determined in a solution

of 0.01N aqueous hydrochloric acid.

• Absorption maxima is approximately at 212nm, 285nm

and 362nm .

• Molar absorptivity of 1% drug solution in a 1cm cell

is 642, 555 and 617 for the wavelengths of 212nm,

285nm and 362nm respectively.

4. High Performance Liquid Chromatography :Principle :• Amiloride is determined by Rp-HPLC method• Where stationary phase is non-polar and mobile phase is polar.

Procedure : Column : C-2 ( 300mm X 4.6 mm i.d)Adsorbent : Silica Gel GParticle size of adsorbent : 10μmTemp : AmbientMobile Phase : 85% aqueous 0.01Msodium hexane sulphonate in acetonotrileFlow rate : 2ml/minRetention time : 2minsDetection : By UV at 280nm.

5. IR spectroscopy :

Principle :

For any molecule to absorb IR radiation it should posses,

• Dipole moment

• Applied frequency of IR must be equal to natural frequency of

radiation.

IR spectra is nothing but a fingerprint of a molecule.

Procedure :1) IR spectrum in a KBr pellet was obtained using a

Fourier-transform (FT IR ) spectrometer.

2) IR spectrum obtained in a mineral oil called mull ( nujol )

IR spectral assignments :

Frequency ( cm-1 ) Assignments3250 -3500 N-H stretch ( NH2)

3150 N-H stretch (NH)

1680 C=O stretch

1640 H2 deformation mode

1600 H2 deformation mode

1240 N- (C6H6) stretch

770 C-H out of plane mode

REFERENCES :

• Text book of Pharmacology by P.D Seth. Pg 292 – 303.

• Analytical Profiles of Drug Substances by Klaus Florey, vol 15, Pg 1 – 30.

• Clinical Pharmacology by P.N.Bennett and M.J.Brown. 9th edition, Pg 529 – 547.

• Modern Pharmacology by Charles.R.Craig and Robert.E.Stitzel,3rd edition, pg 270 – 270.

• The Pharmacological basis of Therapeutics by Goodmann and Gilman, 8th edition, vol 1, pg 708 – 730.