DEVELOPMENT & VALIDATION OF

STABILITY-INDICATING METHOD FOR

SIMULTANEOUS ESTIMATION OF AMLODIPINE
& ENALAPRIL

GUIDE: PREPARED BY:

PROF.(Mrs.) S. J. RAJPUT Bhavik Solanki

Pharmacy Department
Faculty of Technology and Engineering
The M.S. University of Baroda
Vadodara – 390 001
CHROMATOGRAPHY
Chromatographic method must have essentially,
 Stationary phase
 Mobile phase
 Column (support for stationary phase)
 Detection on detecting agent.
 All chromatographic methods involve
modifications in these basic components.
REVERSED-PHASE HPLC
 Principle: Partition of analytes between mobile phase
and stagnant phase inside the pore space + adsorption
on the surface of bonded phase.
 Stationary Phase: Hydrophobic surfaces of moieties
bonded on silica (C18, C8, C5, Phenyl, CN)
 Mobile phase: Methanol or Acetonitrile and Water.
 Applications: ~80% of all separations done on RP
HPLC.
REVERSED PHASE

 80% Octadecylsilica (ODS, C18)

 10% Octylsilica (C8)
 5% Butylsilica (C4)
 3% Phenyl
 2% Cyano (CN)
REVERSED PHASE - SEPARATION PRINCIPLE

 Nonpolar (nonspecific) interactions of analyte with

hydrophobic adsorbent surface (-C18, C8, Phenyl, C4).

 Different sorption affinities between analytes results in

their separation.
• More polar analytes retained less
• Analytes with larger hydrophobic part are
retained longer
• Almost no separation of structural isomers
 REVERSED PHASE - SEPARATION PRINCIPLE
 Nonspecific (hydrophobic) interactions are at least ten
times weaker than polar.

 Small differences in component molecular

structure could have a significant effect in their
retention.

Reversed Phase Normal Phase

Octane, k’ = 4.3 Octanol, k’ = 3.5
Nonane, k’ = 7.8 Nonanol, k’ = 3.3
DRUG PROFILE
SR. NO. PARAMETER AMLODIPINE BESYLATE ENALAPRIL MALEATE

1 STRUCTURE

ANGIOTENSIN CONVERTING
2. CATEGORY CALCIUM CHANNEL BLOCKER
ENZYME (ACE) INHIBITOR
SLIGHTLY SOLUBLE IN WATER,
SLIGHTLY SOLUBLE IN
PROPANOL; FREELY SOLUBLE
3. SOLUBILITY WATER, FREELY SOLUBLE IN
IN METHANOL; SPARINGLY
METHANOL
SOLUBLE IN ETHANOL
MOLECULAR
4. C20H25ClN2O5•C6H6O3S C20H28N2O5.C4H4O4
FORMULA
5. MOLECULAR WEIGHT 567.05 gm/mol 492.53 gm/mol
WHITE TO PALE YELLOW WHITE TO OFF WHITE
6. APPEARANCE
CRYSTALLINE POWDER CRYSTALLINE POWDER
7. DOSAGE 2.5 MG ONCE DAILY 2.5-5MG/DAY
 OPTIMIZATION OF MOBILE PHASE
AMLODIPINE ENALAPRIL
Mob. Phase Flow
Sr. Inj.Vol
Temp. pH rate Column
No. (ratio) (µl)
(ml/min) Rt Peak Rt Peak

Buff:ACN Not
1. 50 2.2 2 10 Hy C8 150 1.43 11.26 Broad
(75:25)7 Clear

Buff:ACN Fronti Broad

2. 50 3.0 2 10 Hy C8 150 1.78 6.96
(75:25)7 -ng

Buff:ACN Not Not

3. 30 3.0 1 10 Int C8 250 5.37 7.04
(75:25)7 Good Good

Buff:MeOH Not
4. 25 3.0 1 10 Int C8 250 5.88 8.55 -
(35:65)7 good

Buff:MeOH Not
5. 25 2.5 1 10 Zor C8 250 5.05 6.89 Tailing
(35:65)7 Good

Buff:MeOH
6. 50 2.5 1 10 Hy C8 250 5.55 - 11.82 Tailing
(45:55)7
 OPTIMIZATION OF MOBILE PHASE
AMLODIPINE ENALAPRIL
Mob. Phase Flow
Sr. Inj.Vol
Temp. pH rate Column
No. (ratio) (µl)
(ml/min) Rt Peak Rt Peak

Buff:MeOH
7. 50 2.5 1.5 10 Hy C8 250 3.94 - 8.30 Tailing
(45:55)7

Buff:MeOH
8. 50 2.5 1.5 10 Hy C8 250 4.29 - 11.56 Tailing
(45:55)2

Buff:MeOH Fronti
9. 50 2.5 2.0 10 Hy C8 250 3.35 7.88 Tailing
(45:55)7 -ng

Buff:MeOH
10. 50 2.5 1.5 10 Hy C8 150 2.39 - 4.49 -
(45:55)7

Buff:MeOH
11. 50 2.2 1.5 10 Hy C8 150 3.96 - 8.19 -
(50:50)7

Buff:MeOH
12. 50 2.2 1.5 10 Hy C8 150 3.30 - 9.35 -
(55:45)7
OPTIMIZED HPLC PARAMETERS

Column BDS Hypersil, C8 , 150mm×4.6mm; 5µ

Mobile
Buffer : Methanol (55:45)
Phase
Flow rate 1.5ml/min
Retention
Amlodipine 3.30min, Enalapril ~ 9.35min
Time
Injection
10 µL
volume

Detection UV at 215nm
STANDARD PREPARATION
35 mg Amlodipine Besylate + 100 mg of Enalapril Maleate in
100ml of Methanol.

Sonicate for 5 min, take 5 ml & dilute to 25 ml Mobile phase.

SAMPLE PREPARATION
Take 5 intact capsule in 100 ml of Mobile phase.

Sonicate for 30 min & Dilute to 25ml with Mobile phase.

LINEARITY CURVE OF ENALAPRIL MALEATE

Concentration Area
(ng/ul)
linearity curve of enalapril
40 325.911
3000
y = 8.0241x + 3.3869
2500 100 814.72064
R2 = 0.9993
2000
1500 160 1284.3731
area

1000
500
0 200 1612.23462
0 50 100 150 200 250 300 350
concentration(ng/ul) 240 1892.62964

300 2435.50319
LINEARITY CURVE OF AMLODIPINE BESYLATE

Concentration Area
(ng/ul)
linearity curve of amlodipine besylate
14 157.86616
1500
y = 11.391x - 3.8895 35 398.53259
1000 R2 = 0.9992

56 631.1651
area

500

0 70 795.15222
0 20 40 60 80 100 120
concentration(ng/ul) 84 934.50269

105 1205.80298
 DEGRADATION OF ENALAPRIL MALEATE
AS HEAT &
SET NO. ACID ALKALI PEROXIDE UV THERMAL
SUCH MOIST

%ASSAY 98.7% 99.3% 77.2% 98.0% 100% 91.6% 100.3%

%DEG. 0.0% 0.7% 21.8% 2% 0.0% 7.2% 0.0%

DEGRADATION OF AMLODIPINE BESYLATE

AS HEAT &
SET NO. ACID ALKALI PEROXIDE UV THERMAL
SUCH MOIST

%ASSAY 93.7% 90.3% 72.9% 79.1% 90.5% 86.1% 91.1%

%DEG. 0.0% 3.6% 22.2% 15.6% 0.0% 3.4% 2.8%

SUMMARY OF VALIDATION PARAMETERS

VALIDATION
AMLODIPINE ENALAPRIL
PARAMETER

LINEARITY R2 = 0.9981 R2 = 0.9984

RANGE 14-105 ng/ul 40-300 ng/ul

ACCURACY 100.63 ± 0.3 100.53 ± 0.5

PRECISION 0.3 0.6

LOD 0.153 0.2242

LOQ 0.459 0.67

ROBUSTNESS % RSD = 0.094 % RSD = 0.093

CONCLUSION

Hence we can conclude that the developed

methods is simple and rapid. Moreover the
methods is quite sensitive to determine
microgram quantities of drugs and economic too.