This document describes the development and validation of an RP-HPLC method for the simultaneous determination of ramipril and amlodipine in tablets. The method utilizes a gradient elution with a C18 column, mobile phase of acetonitrile and sodium perchlorate buffer, and UV detection. The method was validated per ICH guidelines and showed good linearity, accuracy, precision, specificity and robustness. Forced degradation studies demonstrated the method can separate ramipril, amlodipine and their degradation products. The method was successfully applied to determine the content of ramipril and amlodipine in three tablet batches.

1. A PRESENTATION ON :
DEVELOPMENT AND VALIDATION OF AN RP-HPLC
METHOD FOR SIMULTANEOUS DETERMINATION OF
RAMIPRIL AND AMLODIPINE IN TABLETS
1
PRESENTED BY:
AMPATI RAHUL
{M PHARMACY:PHARMACEUTICAL ANALYSIS}

2. INTRODUCTION:
RAMIPRIL: ACE inhibitor, anti hypertensive
2
ANGIOTENSINOGEN
ANGIOTENSIN-1
ANGIOTENSIN-2
AT1 RECEPTOR
ALDOSTERONE
SALT AND WATER
RETENTION
INCREASES BP
RENIN
ACE
RAMIPR
IL
-
-
-
-
-

3. AMLODIPINE: It is the drug that blocks L-type of voltage gated
calcium channel present in blood vessels and heart
3

4. WHY TO GIVE DRUG IN COMBINATION…..?
4

5.  Large majority of hypertensives ultimately require drug
combination to decrease the damage of heart, brain, kidney,
etc.,
 Fixed dose combinations of drugs with complementary
properties offer the advantages of simplicity, tolerability,
convenience and cost effectiveness, as well as the
compliance.
 The combination therapy of ACEI and CCB has proved to
be effective.
 So the combination of RP and AL would also be good
therapeutic option. 5

6. DRUG PROFILE
6
RAMIPRIL AMLODIPINE
STUCTURE
CHEMICAL
NAME
[(2S, 3aS, 6aS)-1-[(S)-2-
[[(S)-1-
(ethoxycarbonyl)-3-
phenylpropyl] amino]
propanoyl] octahydro
cyclopenta [b] pyrrole-2-
carboxylic acid
[3-Ethyl5-methyl(4RS)-2-[(2-
aminoethoxy)
methyl]-4-(2-chlorophenyl)-
6-methyl-1,4-
dihydropyridine-3,5-
dicarboxylate
SIDE
EFFECTS
Hypotension, cough Peripheral edema

7. 7
Chemical structure of impurities:
Ramipril impurities A (II), B (III), C (IV), D
(V)
Amlodipine impurity D

8. OBJECTIVE OF WORK:
 To develop HPLC method for determination of Ramipril and
Amlodipine in the same tablet dosage form.
 To validate the developed methods as per the ICH guidelines.
 To study stability indicating method of drug.
8

9. PLAN OF WORK:
9
Literature survey
Selection and procurement of drug
study of physical properties of drug
Optimization of analytical method
validation of developmental method
Forced degradation studies
Result and discussion
Summary and conclusion

10. REAGENTS AND CHEMICALS
SL
NO
NAME OF THE CHEMICALS MAKE
1 RP API Green syn co., ltd
2 AL besylate API weihai disu pharmc.Co.,ltd
3 Tablet exepients Chengdu haisco pharmaceutical
co.,ltd
4 RS of AL beyslate National institute for control of
pharmaceutical products
5 RS of RP European directorate
6 Impurity standards of RP and AL European directorate
7 HPLC-grade acetonitrile Honey well
8 HPLC-grade triethylamine Kermel chemical reagents
company
10

11. HPLC INSTRUMENTS AND ANALYTICAL
CONDITION:
 Analysis was performed on a chromatographic system of
shimadzu prominence consisting of AT liquid pump and
PDA detector with manual 20 µl sample injection loop.
 Chromatographic seperation was achieved on Inertsil
ODS -3 (250mm * 4.0mm , 3µm) column.
 The column temperature was maintained at 55°C and
flow rate was 1.0mL/min.
 The detection wavelength was set at 210nm. 11

12.  Cont’d….
 Data acquisition was made with shimadzu LC solution
software.
 Mobile phase A consisted of 60mM sodium perchlorate
buffer (containing 7.2mM triethylamine)-
acetonitrile(60:40,v/v)
 Mobile phase B was 60mM sodium perchlorate buffer
(containing 7.2mM triethylamine)- acetonitrile (20:80,v/v).
 The apparent pH of the mobile phase was adjusted to 2.6
with phosphoric acid. 12

13. Sample preparation:
13
Twenty tablets are powdered
25 mg of RP and 50 mg of AL was weighed and dissolved in mobile phase A
Ultra sonicated for 15mins
The mixture was centrifuged at 10000 rpm for 10mins.
The supernatant was separated and transferred into HPLC instrument to be
analyzed

14.  Samples were subjected to stressed conditions of light, heat,
acid, base and oxidation.
 In the stress condition, all the solutions were prepared by
weighing 4-tablets equivalent mass of sample powders
containing about 10mg RP or 20mg AL pure API.
14

15. METHOD DEVELOPMENT
15
Gradient HPLC method was adopted
 To get a shorter run time
 Higher sensitivity.
 Stainless steel column(250mm*4.0mm, 3µm) was used (ph.
Eur.).
 Mobile phase A and B all consisted of acetonitrile, Sodium
perchlorate buffer and 1mL trimethylamine with different
pH values adjusted to 3.6 and 2.6 with phosphoric acid.

16.  As RP and AL had N-H groups as basic nitrogen centers,
the amount of perchlorate and the pH of M.P would affect
the peak shape, resolution and symmetry.
 The results shows that the retention time of RP and AL
were lengthened when the amount of perchlorate increased.
 The RT of RP would be shortened while the RT of AL has
no significant change when the pH was decreased.
 Column diameter of 3µm was chosen
 Column temperature of 60°C, 55°C and 50°C were studied
and 55°C was finally chosen. 16

17. 17
Gradient program proposed for the analysis of AL (Amlodipine), RP
(Ramipril) and their related substances

18. 18
Chromatograms of sample solution (1mg/mL RP and 2mg/mL AL solution using
the final optimized mehtod).1.Benzene sulfonic acid, 2. Unknown 1, 3.Unknown 2,
4.Amlodipine impurity D, 5.Unknown 3, 6.Ramipril impurity B, 7.Unknown 4,
8.Ramipril impurity C, 9. Unknown 5, 10.Unknown 6, 11.Ramipril impurity D,
12.Unknown 7, and 13.Unknown 8.

19. METHOD VALIDATION:
VALIDAT
ED WITH
SYSTEM
SUITABILIT
Y
SPECIFICTY
LINEARITY
RANGE
ACCURACYPRECISION
LOD AND LOQ
STABILITY
ROBUSTNESS
19

20. System suitability test:
 The solution containing 0.1mg/mL of RP, 0.2mg/mL of AL
besylate and each 0.1 mg/mL of five major impurities was
prepared by mobile phase A.
 System suitability was determined by six replicate injections of
the system suitability solution.
 The resolutions among RP, AL and the closest eluting peaks were
bigger than 2 which indicating that this method was reliable for
the quantitation of RP and AL
20

21. Acceptance criteria:
21
Less than 2% RSD
for peak area
Greater than 3000
column plates
Less than 1.5 of the
USP tailing factor
Greater than 1.5 of
the resolution

22. 22
Chromatogram of the system suitability test solution. 1. Benzene sulfonic acid, 2.
Ramipril impurity A, 3. Amlodipine impurity D, 4. Ramipril impurity B, 5. Ramipril
impurity C, and 6. Ramipril impurity D

23. Specificity :
 The selectivity of the method was confirmed by observing
potential interference caused by excipients of tablet
formulations and degradation products .
 The chromatogram of the tablet excipients shows that there
were no interference of peaks to the determination of Rp and
AL.
23

24. Linearity and range:
 A standard stock solution containing 0.25mg/mL of RP and
0.5mg/mL of AL besylate was prepared by mobile phase A
Dilute with the same solvent to yield solution at different
concentrations
These solutions were protected from light using aluminium foil
Stored at 4ºC in the refrigerator
24

25.  The linearity was checked by analyzing six working
solution of RP over the concentration range 0.01 – 0.25
mg/mL and 0.014 – 0.36 mg/mL for AL
 Results :
25
RP AL
y=44164x + 303.8 y= 37963x - 597
r2= 09998 r2= 0.9997

26. Accuracy and precision:
 Known amount of each standard powder was added to
blank sample
 Mixed , extracted and subsequently dilute to yield three
different concentrations of each drug
 RP: 0.08, 0.10 and 0.12 mg/mL
 AL: 0.16, 0.20 and 0.24 mg/mL
26

27. 27
Accuracy results [Recovery (%)] for the determination of AL
(Amlodipine) and RP (Ramipril).

28.  Repeatability or intra – day precision was investigated by
injecting six replicate sample solution on the same day.
 Inter day precision was assessed by analysis newly prepare
sample solutions in triplicate over three consecutive days.
28
RP AL
RSD% RSD%
Intra day 0.46 0.55
Inter day 1.60 1.80

29. LOQ and LOD:
 The LOQs for RP and AL corresponding to a signal-to-noise
ratio of 10 were 0.2µg/mL and 0.07 µg/mL.
 The LODs corresponding to a signal to noise ratio of 3 were
0.06µg/mL and 0.2 µg/mL.
 The resultant RSD values for these studies were < 3.5%
29

30. Stability of solutions and robustness:
 The stability of the standard stock solutions was determined by
quantitatively determining each drug in different time
comparing to the response obtained for freshly prepared
standard solutions.
 The stability of sample solution was tested for every 2h
interval upto 12h.
 The RSD values of RP and AL were 1.2% and 1.0%,
respectively.
30

31. Robustness :
 The robustness of the method was investigated by a small
variety of conditions including changes of pH of the eluent,
flow rate and column temperature .
 The assay results for RP and AL by three different analysts
in the same laboratory were also investigated.
 The RSD values did not exceed 2.5%.
 The degree of reproducibility of the results obtained as a
result of small deliberate variations in the method
parameters and by changing analytical operators
demonstrated that the method was robust.
31

32. 32
Chromatographic parameter setting applied in the
robustness investigation

33. FORCED DEGRADATION STUDIES
Degradation profile under different stress condition:
1. Acid Degradation (HCL)
2. Alkali Degradation (NaOH)
3. Oxidation Degradation (H2O2)
4. Thermal Degradation
5. Photo Degradation 33

34. 34
 Acid Degradation: It was conducted using 2mL of 0.1M
hydrochloric acid

35.  Alkali Degradation: It was carried out in 2mL of 0.1M
sodium hydroxide.
35

36.  Oxidation Degradation: It was performed by adding 2mL of
3% H2O2 and kept in water bath for 5min, and then diluted
with mobile phase A.
36

37.  Thermal Degradation: For the temperature stress study,
compound RPAL tablets, RP and AL besylate API were exposed
to dry heat of 60°C in a convention oven for 10 days.
37

38.  Photo Degradation : For photo stability studies composed
RPAL tablets , RP and AL besylate API were exposed t 4500
lx light in a light cabinet for 10days.
38

39.  All stressed samples were compare with an un-stressed
sample solution.
 The peak purity indices for RP and AL in stressed solutions
were found to be better ( purity angle < purity threshold)
 And they also evidence the ability of the method to access
unequivocally the analytes of interest in the presence of
potential interference.
39

40.  Origination of related substance:
 The origination of the related substances in tablets was
investigated.
 The peaks numbered 6, 8, 11 as defined in the chromatograms of
sample solution( RL impurity B, C, D respectively, according to
the RRT) would same from RP API.
 In addition to the known impurities, the unknown impurities
peaks numbered 2, 9, 10, 12, 13 originated in RP API
 And the peaks numbered 1 ( benzoic acid), 3, 4(Amlodipine
impurity D), 5 and 7 would originate in AL besylate API. 40

41. 41
Chromatograms of RP (Ramipril) API and AL (Amlodipine) besylate API .

42. Assay of AL and RP in tablets:
 Three batches of compound tablets were analysed using
the developed method.
42

43. Conclusion :
 A gradient LC method has been developed and validated for
the analysis of RP and AL in tablet dosage forms.
 The results of the stress testing revealed that the method was
specific and selective.
 The proposed method has the ability to separate the two main
components from their degradation products, related
substances found in tablet dosage forms and the tablet
excipients.
43

44.  Therefore, the chromatographic method can be used to
analyze samples obtained during accelerated stability
experiments and routine assay of RP and AL in combined
tablet dosage forms.
 In addition, the procedure can be further applied for the
detection and determination of the related substances in
tablets
44

45. 45