about medical testing and assay and application of hplc

1. MATER OF PHARMACY
IN
PHARMACEUTICAL CHEMISTRY
Submitted by:
Vijendra singh
Supervised by: Co-supervised by:
Dr. Amardeep Ankalgi Mr. Ketan Patel
Reader and Head Head, Molecule lab
Dept. of Pharm. Chemistry Ahemadabad(Guj.)
BHUPAL NABLES’ COLLEGE OF PHARMACY
UDAIPUR-313002, RAJASTHAN
2014
METHOS DEVELOPMENT AND VALIDATION OF ATORVASTATIN AND
OLMESARTAN TABLET BY USING U.V AND RP-H.P.L.C

2. INTRODUCTION
 Pharmaceutical analysis is a branch of practical chemistry that involves a series
of process for identification, determination, quantification and purification of
all active entity, pharmaceutical excipient, separation of the component of a
solution of a mixture or determination of structure of chemical compounds.
 Analytical chemistry is the study of the separation identification and
quantification, of the chemical components of natural and artificial materials.
These techniques are more accurate, preside sensitives, selective and less time
consuming.
 Analytical chemistry can be divide in two general areas of analysis
a) Quantitative: Determines the amount of and or more these components.
b) Qualitative: It gives an indication of the identity of the chemical species.
 Analytical methods are generally classified into two categories.
 Instrumental:- Electroanalytical, spectroanalytical, chromatographic.
 Non-instrumental:- Animal assay, Gravimetric, titrimetry.
 Method development.
 Method validation.

3. Drug profile :- Atorvastatin
S.NO PARAMETER ATORVASTATIN
1 IUPAC Name (3R,5R)-7-[2-(4-fluorophenyl)-3-phenyl-4-
(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-
dihydroxyheptanoic acid
2 Molecular formula C33H35FN2O5
3 Molecular weight 558.639803 g/mol
4 Categories Antihypertensive Agent
5 Solubility Methanol
6 pKa 4.46
7 Half life 14 h
8 Therapeutic use The primary use of atorvastatin is for the treatment
of dyslipidemia and prevention of cardiovascular
disease.

4. Drug profile :- Olmesartan
S.NO PARAMETER OLMESARTAN
1 IUPAC Name (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl
5-(2-hydroxypropan-2-yl)-2-propyl-3-[[4-[2-(2H-
tetrazol-5-yl)phenyl]
phenyl]methyl]imidazole-4-carboxylate
2 Molecular formula C29H30N6O6
3 Molecular weight 558.585 g/mol
4 Categories Antihypertensive Agent
5 Solubility methanol
6 pKa Strong acidic-0.91,stong basic-5.57
7 Half life 13 h
8 Therapeutic use The primary use of olmesartan is for the treatment
of the hypertension and also in first line agent in
diabetic nephropathy.

5. LITERATURE REVIEW
 Reddy R , et. al. (2013) . Reported simple, rapid and accurate UV Spectroscopic
(Simultaneous Equation method and First order derivative method) and an isocratic RP –
HPLC methods which showed excellent sensitivity, reproducibility, accuracy, and
repeatability.
 Nagavalli, D. et. al. (2011). Reported two simple, precise, accurate, specific and
reproducible Spectrophotometric methods which have been developed for the
simultaneous estimation of Olmesartan medoxomil (OLM) and Atorvastatin calcium
(ATV) in combined tablet dosage form using simultaneous equation and first order
 Delhiraj N, et. al. (2013). Reported two new, rapid, precise, accurate and specific
chromatographic methods for the simultaneous determination of Olmesartan
Medoximil, Amlodipine Besylate and Hydrochlorothiazide in combined
pharmaceutical dosage forms.
 Mhaske R.A. et. al. (2011). Carried out a simple, precise and stability-indicating
HPLC which method was developed and validated for the simultaneous
determination of anti-hypertensive drugs Atorvastatin Calcium, Olmesartan
Medoximil, Candesartan, diuretics Hydrochlorothiazide and Chlorthalidone.
 Baldania S.L , et. al. (2012). Reported a new, simple, accurate, and precise high-
performance thin-layer chromatographic (HPTLC) method is described for
simultaneous analysis of Metoprolol Succinate and Olmesartan Medoxomil in
pharmaceutical formulations.

6. PLAN OF WORK
 Literature survey
 Interaction with academicians & resource
persons
 Development of method by U.V and RP-
H.P.L.C.
 Validation of method by U.V and RP-
H.P.L.C
 Recovery studies

7. DEVELOPMENT OF RP-H.P.L.C METHOD
 Selection of the mode.
 Selection of the column.
 Sample preparations.
 Selection of mobile phase solvents.
 Selection of detection wavelength.
 Optimization of developed method.
 Validation of developed method.

8. MATERIALS & METHOD
REAGENTS:
Reagents are HPLC and AR grades used
Equipments:
Digital weighing balance(Analytical).
pH meter.
HPLC water
Chromatography
Software:-Spinchrom
MATERIALS:
Atorvastatin calcium, Olmesartan medoximil are supplied by Molecule
Lab, Ahemdabad.

9. EXPERIMENTAL WORK
Prepration of standard solution:
Preparation of standard solution of Atorvastatin calcium.
Preparation of standard solution of Olmesartan medoximil.
Working standard solution.
Chromatographic method:
Column:-Thermoscientific, BDS hypersil C18, 250 mm × 4.60 mm, 5µ.
Column Temperature : 250C
Wavelength : 235 nm (PDA)
Flow rate : 1ml/min
Injection Volume : 20µl

10. TRAIL:-1
 Mobile phase:- Methanol : water(50:50 v/v) Flow Rate: 1.0 ml/min.
Conclusion:- No peaks found

11. TRAIL:-2
 Mobile phase:-Water : Acn 50:50 pH 5.0 with H3PO4 Flow Rate: 1.0 ml/min.
 Conclusion:-Atorvastatin peak present but Olmesartan peak is absent.

12. TRAIL:-3
 Mobile phase:-Water : Acn 50:50 pH 4.0 with H3PO4 Flow Rate: 1.0 ml/min.
 Conclusion:-In both peaks tailing present.

13. TRAIL:-4
 Mobile phase:-water-ACN-TEA 50-50-0.1 pH 4.0 with H3PO4 Flow Rate: 1.0
ml/min.
 Conclusion:- In Olmesartan’s peak tailing present.

14. TRAIL:-5
 Mobile phase:-ACN-TEA 40-60-0.1 pH 4.0 with H3PO4 Flow Rate: 1.0 ml/min.
 Conclusion:- peaks found satisfactory

15. VALIDATION OF RP-H.P.L.C METHOD
 Linearity of Atorvastatin:-
y = 98.09x - 71.974
R² = 0.9983
0
2000
4000
6000
0 10 20 30 40 50
a
r
e
a
Conc.(ppm)
atorvastatin
Sr. No. % concentration
with respect
concentration
Concentration
ppm(µg/ml)
Peak Area SD %RSD
1 50
15 1449.003
7.015997805 0.848622
2 75
22.5 2055.952
12.43049729 1.034758
3 100
30 2889.334
14.56158269 0.8753
4 125
37.5 3608.044
17.86644789 0.86846
5 150
45 4351.344
24.16238106 0.968481
Correlation Coefficient 0.998
Regression Equation Y = 98.09 x – 71.97

16.  Linearity of olmesartan:-
y = 47.099x - 80.809
R² = 0.9977
0
2000
4000
6000
0 20 40 60 80 100
a
r
e
a
Conc.(ppm)
olmesartan
Sr. No. % Concentration
with respect to
test
concentration
Concentration
ppm(µg/ml)
Peak Area SD %RSD
1 50
30 1387.602
2.480028185 1.098234
2 75
45 1949.108
3.334183453 0.979915
3 100
60 2766.875
4.806526521 1.053007
4 125
75 3455.199
5.328124276 0.961468
5 150
90 4167.018
7.786318784 1.130555
Correlation Coefficient 0.997
Regression Equation Y = 47.09x -80.80

17. PRECISION:-
 Intraday precision:-
ATORVASTATIN
Reading taken in 3hrs interval
Concernatation % is-50%,100%,150%
OLMESARTAN
Reading taken in 3hrs interval
Concernatation % is-50%,100%,150%
Conc.
ppm
Peak Area
Mean*
SD % RSD Conc.
ppm
Peak Area
Mean *
SD % RSD
15 1445.3866
67
14.857241
01
1.0279077
11
30 1385.0666
67
15.562815
69
1.1236149
18
30 2888.3733
33
37.772608
19
1.3077467
43
60 2767.405 38.462726
23
1.3898481
15
45 4370.0993
33
50.139989
4
1.1473420
99
90 4195.0033
33
59.654411
68
1.4220349
05

18.  INTERDAY PRECISION:-
ATORVASTATIN
Reading taken in a day interval
Concentration taken:-
50%100%150%
OLMESARTAN
Reading taken in a day interval
Concentration taken:-50%100%150%
Conc.
Ppm
Peak Area
Mean*
SD % RSD Conc.
ppm
Peak Area
Mean *
SD % RSD
15
1444.9426
67
14.203723
33
0.982995
634
30
1385.77866
7
16.6356711
4
1.20045657
7
30
2868.5823
33
23.723104
19
0.826997
5
60
2753.55533
3
32.9402702
8 1.1962814
45
4311.6553
33
63.435925
6
1.471266
154
90
4136.761
69.9702008
1
1.69142478
4

19. ACCURACY:-
 Accuracy data of Atorvastatin:-
Conc.
Level (%)
Amount added
(mcg/ml)
Amount Found
(mcg/ml)
% Recovery % Recovery
mean*
S.D. %RSD
80
24 24.37609035 101.5670431
101.5613033 0.543804731 0.5345444814
24 24.5045317 102.1022154
24 24.24351634 101.0146514
100
30 30.10018097 100.3339366
100.2154853 0.22533945 0.224854896
30 30.10706873 100.3568958
30 29.98668709 99.95562362
120
36 36.57757694 101.6043804
100.3134717 1.144072548 1.140487179
36 35.9697473 99.91596473
36 35.79219723 99.42277008

20.  Accuracy data of olmesartan:-
Conc.
Level (%)
Amount added
(ppm)
Amount Found
(ppm)
% Recovery % Recovery
mean*
S.D. % RSD
80
48 48.75454098 101.5719604
101.5644458 0.544714732 0.53632423
48 49.01057486 102.1053643
48 48.48768604 101.0160126
100
60 61.05200404 101.7533401
100.690868 0.943560772 0.937086739
60 60.22116604 100.3686101
60 59.97039239 99.95065399
120
72 73.15327108 101.6017654
100.7030525 0.844989244 0.839090001
72 71.94577879 99.92469276
72 72.41954345 100.5826992

21. ROBUSTNESS:-
CONDITION
PEAK AREA MEAN * SD %RSD
ATOR OLME ATOR OLME ATOR OLME
Change in the Mobile Phase Composition(± 2 ml organic Phase)
Change in the -2ml
organic phase
(58:42 v/v)
2884.978 2766.069 34.43744 38.40014 1.193681 1.388257
Change in the + 2
ml organic phase
(62:38 v/v)
2885.172 2764.169 31.33029 31.89391 1.085907 1.153833
Change pH(±0.2 unit)
Change in the -0.2
unit pH
2898.877 2776.078 23.45083 22.5003 0.808963 0.810507
Change in the +0.2
unit pH
2869.378 2760.55 25.00704 43.78793 0.871514 1.586203
Change Flow rate (±0.2 ml/min)
Change in the -
0.2ml/min F.R.
3040.007 2911.643 28.06252 27.65838 0.923107 0.949923
Change in the
+0.2ml/min F.R.
2747.349 2631.29 28.41529 27.82877 1.03428 1.057609

22. SYSTEM SUITABILITY
 System suitability of Atorvastatin and olmesartan:-
Sr. No. Parameters OLME ATOR
1. Avg. peak area of
standard
1665.52 459.25
2. No. of theoretical
plates
6845.8 7013.6
3. Retention time (min) 3.39 ± 0.10 5.70 ± 0.10
4. Asymmetry 1.4 1.4
5. % RSD 1.20 % 1.16 %
6. Resolution - 10.6288

23. REFERENCES
 Willard-Hobart H, Merritt Jr Lynne L, Dean John A (1974) 5th
Ed Instrumental Methods of Analysis. Von Nostrand, University
of Michigan. Page no-375-390
 Davidson AG (2002) Ultraviolet-visible absorption
spectrophotometry. In BeckettAH, Stenlake JB, 4thEd, Practical
Pharmaceutical chemistry. CBS Publishers and distributors,
New Delhi, 275-278.
 Daly FF, Fountain JS, Murray L, Graudins A, Buckley NA
(2008) Guidelines for the management of paracetamol
poisoning in Australia and New Zealandexplanation and
elaboration. A consensus statement from clinical toxicologists
consulting to the Australasian poisons information centres.
Med J Aust 188: 296-301.
 Borne Ronald F (1995) Nonsteroidal Anti-inflammatory Drugs.
In Principles of Medicinal Chemistry,4thEd , William OFoye,
Thomas L Lemke, David A Williams, Williams & Wilkins, page
no- 544-545.
 Chung S, Ben-Menachem E, Sperling MR, Rosenfeld W,
Fountain NB, Benbadis S. et al. Examining the clinical utility of

24. REFERENCES
 Dollery C. Therapeutic drugs .second edn. ChurchillLivingstone,
London UK.1999;C135-C140.
 Pospisilova B and Kubes J. Mercurimetric determination
ofcephalosporin antibiotics. Pharmazie.1998; 43(4):246-248.
 Shaha NJ, Shaha SK, Patel VF, Patel NM. Developmentand
validation of HPTLC method for the estimation of cefuroxime
axetil. Ind J Pharm Sci.2007;69(1):140-142.
 ICH, Q2 (R1) Validation of Analytical Procedure: Text and
Methodology, International Conference on
Harmonization,Geneva, Switzerland; 2005
 Matthews, C.Z. Woolf, E.J. Mazenko, R.S. Haddix-Wiener. H.
Chavez-Eng, C.M. Constanzer, M.L. Doss,G.A. and
Matuszewski. B.K. Determination of efavirenz, a selective non-
nucleoside reverse transcriptase inhibitor, in human plasma
using HPLC with post-column photochemical derivatization and
fluorescence detection. J. Pharmaceut. Biomed. Anal. 2000;
28:925–934.

25. THANK YOU